Tumor specific antibody

ABSTRACT

The present invention provides the amino acid and nucleic acid sequences of heavy chain and light chain complementarity determining regions of a tumor specific antibody. In addition, the invention provides tumor-specific antibodies and immunoconjugates comprising the tumor-specific antibody attached to a toxin or label, and methods and uses thereof. The invention also relates to diagnostic methods and kits using the tumor-specific antibodies of the invention.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 11/570,198filed Jun. 27, 2007, now U.S. Pat. No. 8,383,117, which is a nationalphase entry application of PCT/CA2005/000899 filed Jun. 10, 2005 (whichdesignated the U.S.) which claims the benefit of U.S. provisionalapplication Ser. No. 60/578,291 filed Jun. 10, 2004. All of the priorapplications are incorporated herein by reference in their entirety.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the Sequence Listing “10241-P1214US02”(98,304 bytes), submitted via EFS-WEB and created on Jan. 17, 2013, isherein incorporated by reference.

FIELD OF THE INVENTION

The invention relates to human tumor-specific binding proteins and alluses thereof. In particular, the invention relates to antibodies orantibody fragments specific for antigens or molecules on cancer cellsand to immunoconjugates comprising the binding proteins of theinvention, and methods of use thereof.

BACKGROUND OF THE INVENTION

In the year 2000, an estimated 22 million people were suffering fromcancer worldwide and 6.2 millions deaths were attributed to this classof diseases. Every year, there are over 10 million new cases and thisestimate is expected to grow by 50% over the next 15 years (WHO, WorldCancer Report. Bernard W. Stewart and Paul Kleihues, eds. IARC Press,Lyon, 2003). Current cancer treatments are limited to invasive surgery,radiation therapy and chemotherapy, all of which cause eitherpotentially severe side-effects, non-specific toxicity and/ortraumatizing changes to ones body image and/or quality of life. Cancercan become refractory to chemotherapy reducing further treatment optionsand likelihood of success. The prognosis for some cancer is worse thanfor others and some, like lung or pancreatic cancer are almost alwaysfatal. In addition, some cancers with a relatively high treatmentsuccess rate, such as breast cancer, also have a very high incidencerate and, thus, remain major killers.

For instance, there are over 1 million new cases of breast cancer,worldwide, each year. Treatments consist of minimal to radical surgicalremoval of breast tissue and lymph nodes with radiation and chemotherapyfor metastatic disease. Prognosis for localized disease is relativelygood with a 5 years survival rate of around 50% but once the cancer hasmetastasized, it is incurable with an average survival of around 2years. Despite improving treatment success rates, nearly 400,000 womendie of breast cancer each year, the highest number of deaths to cancerin woman, ahead of deaths to lung cancer. Among the short and long termsurvivors, most will suffer the lifelong trauma of the invasive anddisfiguring surgical treatment.

Another example is liver cancer, with more than half a million new caseseach year and nearly the same number of deaths due to poor treatmentefficacy. Hepatocellular carcinomas represent around 80% of all livercancers and are rarely curable. Five-year survival rate is only about10% and survival after diagnosis often less than 6 months. Althoughsurgical resection of diseased tissue can be effective, it is not anoption for the majority of cases because of the presence of cirrhosis ofthe liver. Hepatocellular carcinomas are largely radiation resistant andresponse to chemotherapy is poor.

Yet another example is that of pancreatic cancer with around 200,000 newcases per year and a very poor prognosis. In fact, the majority ofpatients die within a year of diagnosis and only a few percent ofpatients survive five years. Surgery is the only available treatment butis associated with high morbidity and complication rates because itinvolves not only the resection of at least part of the pancreas, butalso of all of the duodenum, part of the jejunum, bile duct andgallbladder and a distal gastrectomy. In some cases, the spleen andlymph nodes are also removed.

Bladder cancer is the 9th most common cancer worldwide with an estimated330,000 new cases and 130,000 deaths each year. In Europe, this diseaseis the cause of death for approximately 50,000 people each year. Currenttreatment includes the intravesicular delivery of chemotherapy andimmunotherapy with the bacille Calmette-Guerin (BCG) vaccine thatinvolves the additional risk of systemic infection with the tuberculosisbacterium. Despite this aggressive treatment regime, 70% of thesesuperficial papillary tumors will recur over a prolonged clinical coursesome will progress into invasive carcinomas. The high rate of recurrenceof this disease and associated repeated course of treatment makes thisform of cancer one of the most expensive to treat over a patient'slifetime. For patients with recurring disease, the only options are toundergo multiple anesthetic-requiring cystoscopy surgery or major,radical, life-altering surgery (usually cystectomy). Radical cystectomyconsists of excision of the bladder, prostate and seminal vesicle inmales and of the ovaries, uterus, urethra and part of the vagina infemales.

There are many more examples of cancer where current treatments do notmeet the needs of patients either due to their lack of efficacy and/orbecause they have high morbidity rates and severe side-effects. Thoseselected statistics and facts however, illustrate well the need forcancer treatments with better safety and efficacy profiles.

One of the causes for the inadequacy of current cancer treatments istheir lack of selectivity for affected tissues and cells. Surgicalresection always involves the removal of apparently normal tissue as a“safety margin” which can increase morbidity and risk of complications.It also always removes some of the healthy tissue that may beinterspersed with tumor cells and that could potentially maintain orrestore the function of the affected organ or tissue. Radiation andchemotherapy will kill or damage many normal cells due to theirnon-specific mode of action. This can result in serious side-effectssuch as severe nausea, weight loss and reduced stamina, loss of hairetc., as well as increasing the risk of developing secondary cancerlater in life. Treatment with greater selectivity for cancer cells wouldleave normal cells unharmed thus improving outcome, side-effect profileand quality of life.

The selectivity of cancer treatment can be improved by using antibodiesthat are specific for molecules present only or mostly on cancer cells.Such antibodies can be used to modulate the immune system and enhancethe recognition and destruction of the cancer by the patient's ownimmune system. They can also block or alter the function of the targetmolecule and, thus, of the cancer cells. They can also be used to targetdrugs, genes, toxins or other medically relevant molecules to the cancercells. Such antibody-drug complexes are usually referred to asimmunotoxins or immunoconjugates and a number of such compounds havebeen tested in recent year [Kreitman R J (1999) Immunotoxins in cancertherapy. Curr Opin Immunol 11:570-578; Kreitman R J (2000) Immunotoxins.Expert Opin Pharmacother 1:1117-1129; Wahl R L (1994) Experimentalradioimmunotherapy. A brief overview. Cancer 73:989-992; Grossbard M L,Fidias P (1995) Prospects for immunotoxin therapy of non-Hodgkin'slymphoma. Clin Immunol Immunopathol 76:107-114; Jurcic J G, Caron P C,Scheinberg D A (1995) Monoclonal antibody therapy of leukemia andlymphoma. Adv Pharmacol 33:287-314; Lewis J P, DeNardo G L, DeNardo S J(1995) Radioimmunotherapy of lymphoma: a UC Davis experience. Hybridoma14:115-120; Uckun F M, Reaman G H (1995) Immunotoxins for treatment ofleukemia and lymphoma. Leuk Lymphoma 18:195-201; Kreitman R J, Wilson WH, Bergeron K, Raggio M, Stetler-Stevenson M, FitzGerald D J, Pastan I(2001) Efficacy of the anti-CD22 recombinant immunotoxin BL22 inchemotherapy-resistant hairy-cell leukemia. N Engl J Med 345:241-247].Most antibodies tested to date have been raised against known cancermarkers in the form of mouse monoclonal antibodies, sometimes“humanized” through molecular engineering. Unfortunately, their targetscan also be present in significant quantities on a subset of normalcells thus raising the risk of non-specific toxic effects. Furthermore,these antibodies are basically mouse proteins that are being seen by thehuman patient's immune system as foreign proteins. The ensuing immunereaction and antibody response can result in a loss of efficacy or inside-effects.

The inventors have used a different approach in their development ofantibodies for cancer treatment. Instead of immunizing experimentalanimals with cancer cells or isolated cancer cell markers, they havesought out only those markers that are recognized by the patient's ownimmune system or, in other words, that are seen by the immune system asa foreign molecule. This implies that the markers or antigens areusually substantially absent on normal cells and, thus, the risk ofnon-specific toxicity is further reduced. Hybridoma libraries aregenerated from cancer patient-derived lymphocytes and the antibodiesthey secrete are tested for binding to normal and tumor cells. Onlyantibodies showing high selectivity for cancer cells are retained forfurther evaluation and development as a cancer therapeutic or diagnosticagent. One such highly selective antibody is the subject of this patentapplication. In addition to being selective, this antibody is fullycompatible with the patient's immune system by virtue of being afully-human protein. The antibody of the invention can be used fordiagnostic or therapeutic uses or as a basis for engineering otherbinding molecules for the target antigen.

The basic structure of an antibody molecule consists of four proteinchains, two heavy chains and two light chains. These chains areinterconnected by disulfide bonds. Each light chain is comprised of alight chain variable region and a light chain constant region. Eachheavy chain is comprised of a heavy chain variable region and a heavychain constant region. The light chain and heavy chain variable regionscan be further subdivided into framework regions and regions ofhypervariability, termed complementarity determining regions (CDR). Eachlight chain and heavy chain variable region is composed of three CDRsand four framework regions.

CD44 represents a family of cell surface glycoproteins encoded by asingle gene comprising a total of 20 exons. Exons 19 and 20 areexpressed together as the cytoplasmic tail and therefore grouped as“exon 19” by most research groups (Liao et al. J. Immunol. 151:6490-99,1993). The term exon 19 will be used henceforth to designate genomicexons 19 and 20. Structural and functional diversity is achieved byalternative splicing of the messenger RNA involving 10 “variant” exonsidentified as exons 6-15 or, most often, as “variant exons” 1-10(v1-v10). In human, variant exon 1 contains a stop codon and is notusually expressed. The longest potential CD44 variant is thereforeCD44v2-10 (see Naor et al. Adv Cancer Res 71:241-319, 1997 for review ofCD44).

Exons 1-5 and all variant exons are part of the extracellular domain andcontain many potential sites for post-translational modifications. Thetransmembrane domain is highly conserved across species but theintracellular tail can be truncated leading to another type of variant.One such variant comprises variant exons 8-10 but lacks part of exon 19.Changes to the intracellular domain has been shown to change thefunction of CD44, in part with respect to binding and internalization ofhyaluronic acid (HA). CD44 is not only involved in binding to theextracellular molecules but it also has cell signaling properties (seeTurley et al. J Biol Chem 277(7):4589-4592, 2002 for review).

The “standard” CD44 (CD44s), the most commonly expressed form of CD44,contains exons 1-5 and 16-19 and none of the variant exons. Themolecular weight for the core protein is 37-38 kDa but posttranslationalmodification can result in a molecule of 85-95 kDa or more (Drillenburget al., Blood 95(6): 1900, 2000). It binds hyaluronic acid (HA), anextracellular glycosaminoglycan, constitutively and CD44 is oftenreferred to as the HA receptor. It is interesting that the presence ofvariant exons can reduce the binding of HA by CD44 such that CD44variants cannot be said to constitutively bind HA but such binding canbe inducible (reviewed in Naor et al. Adv Cancer Res 71:241-319, 1997).See FIG. 17 for some examples of variants.

CD44E, also called CD44v8-10, contains variant exons 8-10 in addition tothe exons 1-5 and 16-19. Other variants include CD44v3-10, CD44v6,CD44v7-8 and many others. The variant exons are part of theextracellular domain of the CD44.

CD44E can be present on certain normal epithelial cells, particularly bygenerative cells of the basal cell of stratified squamous epithelium andof glandular epithelium (Mackay et al. J Cell Biol 124(1-2):71-82, 1994)and in the fetus at certain stages development. But importantly, it hasbeen shown to be overexpressed on various types of cancer cells. UsingRT-PCR, lida & Bourguignon (J Cell Physiol 162(1):127-133, 1995) andKalish et al. (Frontiers Bioscience 4(a):1-8, 1999) have shown thatCD44E is present in normal breast tissue and is more abundant thanCD44s. They have also shown that CD44, including CD44E and CD44s areoverexpressed, and preferentially located in metastatic breast cancertissues. Miyake et al. (J Urol 167(3):1282-87, 2002) reported thatCD44v8-10 mRNA is strongly expressed in urothelial cancer and can evenbe detected in urinary exfoliated cells of patients with invasive vssuperficial urothelial cancer. The ratio of CD44v8-10 to CD44v10 mRNAincreases in cancer and was shown to have diagnostic value in breast,lung, laryngeal and bladder. The presence of CD44v8-10 was alsoconfirmed by immunohistochemistry with a polyclonal antibody (Okamoto etal. J Natl Cancer Inst 90(4): 307-15, 1997). CD44v8-10 can also beoverexpressed in gallbladder cancer (Yamaguchi et al. Oncol Rep7(3):541-4, 2000), renal cell carcinoma (Hara et al. Urology54(3):562-6, 1999), testicular germ cell tumors (Miyake et al. Am JPathol 152(5): 1157-60, 1998), non-small cell lung carcinomas (Sasaki etal. Int J Oncol 12(3):525-33, 1998), colorectal cancer (Yamaguchi et al.J Clin Oncol 14(4): 1122-27, 1996) and gastric cancer (Yamaguchi et al.Jpn J Cancer Res 86(12): 1166-71, 1995). Overexpression of CD44v8-10 wasalso shown to have diagnostic value for prostate cancer (Martegani etal. Amer J Pathol 154(1): 291-300, 1999).

Alpha-fetoprotein (AFP) is a major serum protein synthesized duringfetal life. Its presence in adults is usually indicative of carcinomas,particularly those of the liver and teratocarcinomas. It is part of thealbuminoid gene family that also comprises serum and alpha albumins andvitamin D-binding protein. AFP comprises 590 amino acids for a molecularweight of about 69-70 kDa and has one site for glycosylation. (Morinagaet al., Proc Natl Acad Sci 80:4604-08, 1983; Mizejewski Exp Biol Med226(5):377-408, 2002). Molecular variants have been studied andidentified in rodents, but in humans there are no reports of variantproteins being detected. A recent report has identified a variant mRNAthat, if expressed, would code for a 65 kDa protein. This protein isexpected to remain in the cytoplasm (Fukusawa et al. J Soc GynecolInvestig May 20, e-publication, 2005).

SUMMARY OF THE INVENTION

The present inventors have prepared human tumor-specific antibodies thatbind to several types of tumor cells including bladder, breast, ovary,prostate and uterus. Importantly, the antibodies do not significantlybind to normal tissue making them suitable candidates for tumor therapy.The inventors have cloned and sequenced the antibodies and determinedthe sequence of the antibody light and heavy chain variable regions andcomplementarity determining regions 1, 2 and 3. Accordingly, theinvention provides isolated light chain complementarity determiningregions 1, 2 and 3, comprising the amino acid sequences SGDNLGNKYVC (SEQID NO:1), EDTKRPS (SEQ ID NO:2) and QAWDSRTEI (SEQ ID NO:3),respectively; and isolated heavy chain complementarity determiningregions 1, 2 and 3, comprising the amino acid sequences GDEYYWS (SEQ IDNO:4), YMSYRGSSYYSPSLQS (SEQ ID NO:5) and KYCGGDCRSGFDI (SEQ ID NO:6),respectively.

The invention also provides isolated nucleic acid sequences encodinglight chain complementarity determining regions 1, 2 and/or 3,comprising the amino acid sequences SGDNLGNKYVC (SEQ ID NO:1), EDTKRPS(SEQ ID NO:2) and QAWDSRTEI (SEQ ID NO:3), respectively; and isolatednucleic acid sequences encoding heavy chain complementarity determiningregions 1, 2 and/or 3, comprising the amino acid sequences GDEYYWS (SEQID NO:4), YMSYRGSSYYSPSLQS (SEQ ID NO:5) and KYCGGDCRSGFDI (SEQ IDNO:6), respectively.

Additional aspects of the invention are isolated light chain variableregions comprising light chain complementarity determining regions 1, 2and/or 3 of the invention (SEQ ID NOS:1-3), and isolated heavy chainvariable regions comprising heavy chain complementarity determiningregions 1, 2 and/or 3 of the invention (SEQ ID NOS:4-6). In oneembodiment, the light chain variable region comprises the amino acidsequence shown in FIG. 1 (SEQ ID NO:7). In another embodiment, the heavychain variable region comprises the amino acid sequence shown in FIG. 2(SEQ ID NO:9).

The invention also provides an isolated nucleic acid sequence encodingthe light chain variable region of the invention, and an isolatednucleic acid sequence encoding the heavy chain variable region of theinvention. In one embodiment, the light chain variable region comprisesthe nucleic acid sequence shown in FIG. 1 (SEQ ID NO: 8). In anotherembodiment, the heavy chain variable region comprises the nucleic acidsequence shown in FIG. 2 (SEQ ID NO: 10).

Another aspect of the invention is a binding protein, preferably anantibody or antibody fragment, that comprises at least one light chaincomplementarity determining region of the invention (i.e. one or more ofthe SEQ ID NOS:1-3) and/or at least one heavy chain complementaritydetermining region of the invention (i.e. one or more of SEQ ID NO:4-6).The invention also provides a binding protein, preferably an antibody orantibody fragment that comprises the light chain variable regions of theinvention and/or the heavy chain variable regions of the invention.

The inventors have also identified the antigen that binds to the bindingproteins of the invention. Accordingly, the invention provides thebinding protein of the invention that binds to a protein comprising the5-v8 interface of CD44E, the v8 exon of CD44 or amino acid sequenceATNMDSSHSIT. The invention also provides a binding protein of theinvention that binds to CD44E; alpha-fetoprotein; a protein having amolecular weight between 47-53 kDa and an isoelectric point between5.2-5.5, preferably 5.4; a protein having a molecular weight between48-54 kDa and an isoelectric point between 5.1-5.4, preferably 5.2; or aprotein comprising the amino acid sequence 107 to 487 of AFP (SEQ IDNO:14), 107 to 590 of AFP (SEQ ID NO:15) or 107 to 609 of AFP (SEQ IDNO:16).

In addition, the invention provides compositions comprising the bindingproteins of the invention, such as antibodies and antibody fragments,with a pharmaceutically acceptable excipient, carrier, buffer orstabilizer.

Another aspect of the invention is an immunoconjugate comprising (1)binding protein of the invention, preferably an antibody or antibodyfragment that binds to an antigen or molecule on or in a cancer cell,attached to (2) an effector molecule. A further aspect of the inventionis an immunoconjugate comprising (1) binding protein of the invention,preferably an antibody or antibody fragment that binds to an antigen ormolecule that is internalized by a cancer cell, attached to (2) aneffector molecule. In a preferred embodiment, the effector molecule is(i) a label, which can generate a detectable signal, directly orindirectly, or (ii) a cancer therapeutic agent, which is eithercytotoxic, cytostatic or otherwise prevents or reduces the ability ofthe cancer cells to divide and/or metastasize. Preferably, the cancertherapeutic agent is a toxin.

The invention also provides compositions comprising the immunoconjugateof the invention and uses of the immunoconjugate for the manufacture ofa medicament for treating or preventing cancer, and diagnostic purposes.In addition, the invention provides methods of treating or preventingcancer using the immunoconjugate of the invention and related kits.

A further aspect of the invention is a method of diagnosing cancer in amammal comprising the steps of:

-   -   (1) contacting a test sample taken from said mammal with a        binding protein of the invention that binds to an antigen on or        in the cancer cell under conditions that permit the formulation        of a binding protein-antigen complex;    -   (2) measuring the amount of binding protein-antigen complex in        the test sample; and    -   (3) comparing the amount of binding protein-antigen complex in        the test sample to a control.

The invention also includes a method of diagnosing cancer in a mammalcomprising the steps of:

-   -   (1) contacting a test sample taken from said mammal with a        binding protein of the invention that binds specifically to        alpha-fetoprotein or a variant thereof under conditions that        permit the formulation of a binding protein-alpha-fetoprotein        complex;    -   (2) measuring the amount of binding protein-alpha-fetoprotein        complex in the test sample; and    -   (3) comparing the amount of binding protein-alpha-fetoprotein        complex in the test sample to a control.

Another aspect of the invention is a diagnostic agent comprising theimmunoconjugate of the invention, wherein the effector molecule is alabel, which can generate a detectable signal, directly or indirectly.

The invention also includes an isolated protein that can specificallybind with one of the binding proteins of the invention, nucleic acidsequences and uses thereof.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIG. 1 is the nucleic acid and amino acid sequence of the light chainvariable region of VB1-008.

FIG. 2 is the nucleic acid and amino acid sequence of the heavy chainvariable region of VB1-008.

FIG. 3 is SKBR-3 (400× mag) fixed-cell pellet stained with VB1-008 (A)and the isotype control antibody 4B5 (B). Notice prominent membranestaining (arrow).

FIG. 4 are representative photographs of immunohistochemical staining ofnormal testis with VB1-008 and the isotype control antibody 4B5. (A)Sample 925 testes tissue (400× mag) stained with VB1-008. Membranestaining in mature sperm cells is indicated by an arrow. (B) Sample 925testes tissue (400× mag) stained with IgG isotype control 4B5. Noticeabsence of staining. Arrow points to mature sperm cell for contrast tostaining with VB1-008 in (A).

FIG. 5 shows Sample 3427A1 breast adenocarcinoma (400×) stained withVB1-008 and IgG isotype control 4B5. Notice staining of cell membrane oftumor cells, especially of cells in contact with the extracellularmatrix (white arrow). Cells close to the center of the tumor showprimarily cytoplasmic staining (black arrow). Arrow points to unstainedtumor cells. Tumor cells are stained with VB1-008.

FIG. 6 shows Sample 946 B1 bladder carcinoma (400×) stained with VB1-008(A) and IgG isotype control 4B5 (B). Arrows indicate membrane stainingof the tumor cells with VB1-008 (A) but not with the control antibody(B).

FIG. 7 shows sample 4036A2 uterus carcinoma (200× mag) stained withVB1-008 and the IgG control antibody 4B5. Notice membrane staining(arrow) with VB1-008 (A & C) but not with the control antibody (B).Higher magnification of uterus carcinoma (600×) shows membrane staining(C).

FIG. 8 is a demonstration of antibody cell surface binding afterincubation of A-375 cells at different temperatures as determined byflow cytometry. Fluorescence labeling of A-375 cells after incubation ofcell suspensions at 4° C.: 4B5 (1) and VB1-008 (2) Fluorescence labelingof A-375 cells after warming antibody-bound cells to 37° C.: VB1-008 for60 min (3), for 120 min (4).

FIG. 9 shows confocal microscopy assessment of VB1-008 internalization.A-375 cells were incubated with antibody at 4° C., washed and warmed to37° C. for 60 min. Cells were fixed, permeabilized and labeled withfluorescent-labeled second antibody. Fluorescence labeling of A-375cells after incubation of VB1-008 at 4° C. for 60 min, displayingcircumferential surface distribution of labeling, (60××4) magnification(A). Following incubation of antibody-bound cells at 37° C. for 60 minthe cells show strong intracellular staining by internalized antibody,(60××4) magnification (B).

FIGS. 10A, B and C show a western analysis of immunoprecipitationreactions using VB1-008. FIG. 10A shows the results of the experimentunder non-reducing conditions, while FIGS. 10B and C show the results ofthe experiment under reducing conditions.

FIGS. 11A and B show the presence of two distinct protein spots in thepurified antigen complex, very close in molecular weight. The proteinswere probably not perceived as two bands in 1D-PAGE due to proteinstacking. FIG. 11A represents the western blot profile of the 2D-gel andFIG. 11B represents the Coomassie stained counterpart.

FIGS. 12A and B show the mapping of the peptides obtained and thesequence coverage of the original AFP molecule, Accession #GI|4501989.FIG. 12A shows the mapping of peptides obtained from the 2D gel (SEQ IDNO: 76). The amino acids in bolded font represent the sequences of aminoacids identified from MS analysis. The shaded regions represent thehomology of peptide sequences and thereby depict the sequence coverage.FIG. 12B shows the complete mapping of the peptides obtained from the 1Dand 2D gels (SEQ ID NO: 77). The amino acids in bolded font representthe sequences of amino acids from MS analysis. The shaded regionsrepresent the homology of peptide sequences and thereby detect thesequence coverage. The underlined amino acids were not detected.

FIG. 13 shows immunopurification of the VB1-008 antigen using 1000 [×gof MDA-MB-435S membranes as the source. The purified antigen(s) wasresolved on SDS-PAGE under non-reducing sample conditions. Reducingagents such as DTT or p-mercaptoethanol were avoided so as to preservethe native conformation of the binding antigen(s). The sample wasresolved on two lanes of the gel. One lane (A), was stained for protein;the other (B) was subjected to western blotting and probed with VB1-008,to ensure the presence of the specific antigen. Band “E” from thecoomassie stained portion of the gel was excised and sent for MSanalysis.

FIG. 14 shows the complete mapping of the peptides obtained and thesequence coverage of CD44 molecule, Accession #GI|105583 (SEQ ID NO:78). The amino acids in red font represent the sequences of amino acidsidentified from MS analysis. The shaded regions represent the homologyof peptide sequences and thereby depict the sequence coverage. The aminoacids in underlined area constitute the variable region (v8-v10)characteristic of the isoform3 or CD44E.

FIG. 15A shows the reactivity of VB1-008 to recombinant AFP molecule,commercially available from RDI systems. The recombinant AFP waselectrophoresed, transferred on to nitrocellulose membrane and probedwith VB1-008. The results are clearly indicative of the reactivity ofVB1-008 to AFP.

FIGS. 15B and C are 2D-gel profiles of “B” and “C”, which wereimmunoprecipitates obtained using VB1-008. The gels were transferred tonitrocellulose and probed with anti-CD44 and anti-AFP, bothmouse-monoclonal antibodies respectively.

FIG. 16 is a western analysis under non-reducing conditions. Anti-CD44was used to immunopurify CD44 proteins from MDA-MB-435S cells and thepurified fraction was subjected to SDS-PAGE and WB analysis undernon-reducing conditions. The experiment was performed in three sets andeach set was identical to the other. Each of the sets was probed with 5ng/mL of anti-CD44, anti-AFP and VB1-008. Anti-CD44 and anti-AFP weremouse monoclonal antibodies, whereas, VB1-008 is VBI's human monoclonalantibody.

FIG. 17 is a schematic representation of the distribution of differentexons in the CD44 gene in humans. Alternative splicing in the variableregion results in the creation of a number of isoforms, a few of thereported isoforms are represented schematically in the correspondingfigure.

FIG. 18A depicts the amino acid sequence of CD44E (SEQ ID NOS: 79 & 80).The highlighted portion represents the stretch of 17 amino acids used togenerate peptides 1-3. The negative control peptide is highlighted inthe C-terminal region of the protein. FIG. 18B shows the results of abinding experiment with VB1-008 to peptides 1-3.

FIG. 19A shows the results of a competition study using peptides 1-3against binding of VB1-008. FIG. 19B shows the results of a competitionstudy using peptides 1-3 against a control antibody (anti-EGFR).

FIG. 20 shows the nucleotide sequence of the immunoconjugate VB6-008(SEQ ID NO:11). The sequence of the PelB leader sequence is in lowercase with the initiation codon bolded. The stop codes are in uppercaseand bolded.

FIG. 21 shows the amino acid sequences of the heavy chain and lightchain of the immunoconjugate VB6-0008 (SEQ ID NO: 12 and 13).

FIG. 22 shows the complete VB6-008 construct (SEQ ID NOS: 81, 82 and83).

FIG. 23 shows the VB6-008 unit #1, which includes thePelB-VH-CH-Furin-De-Bouganin (SEQ ID NOS: 84 and 85).

FIG. 24 shows the VB6-008 #2 unit which consists of PelB-VL-CL (SEQ IDNOS: 86 and 87).

FIG. 25 shows the results of an in vitro cytotoxicity experiment usingVB6-008.

FIG. 26 is a depiction of the gamma cassette.

FIG. 27 is a depiction of the assembly of the Fab-bouganin immunotoxin.

DETAILED DESCRIPTION OF THE INVENTION (A) Definitions

The term “administered systemically” as used herein means that theimmunoconjugate and/or other cancer therapeutic may be administeredsystemically in a convenient manner such as by injection (subcutaneous,intravenous, intramuscular, etc.), oral administration, inhalation,transdermal administration or topical application (such as topical creamor ointment, etc.), suppository applications, or means of an implant. Animplant can be of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers.Suppositories generally contain active ingredients in the range of 0.5%to 10% by weight.

The term “antibody” as used herein is intended to include monoclonalantibodies, polyclonal antibodies, and chimeric antibodies. The antibodymay be from recombinant sources and/or produced in transgenic animals.The term “antibody fragment” as used herein is intended to include Fab,Fab′, F(ab′)₂, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, andmultimers thereof and bispecific antibody fragments. Antibodies can befragmented using conventional techniques. For example, F(ab′)₂ fragmentscan be generated by treating the antibody with pepsin. The resultingF(ab′)₂ fragment can be treated to reduce disulfide bridges to produceFab′ fragments. Papain digestion can lead to the formation of Fabfragments. Fab, Fab′ and F(ab′)₂, scFv, dsFv, ds-scFv, dimers,minibodies, diabodies, bispecific antibody fragments and other fragmentscan also be synthesized by recombinant techniques.

The term “antibody or antibody fragment of the invention” as used hereincomprises at least one light chain complementarity determining region ofthe invention (i.e. one or more of SEQ ID NOS:1-3) and/or at least oneheavy chain complementarity determining region of the invention (i.e.one or more of SEQ ID NOS:4-6). Preferably, the antibody or antibodyfragment comprises the light chain CDR sequences (SEQ ID NOS:1-3) and/orthe heavy chain CDR sequences (SEQ ID NOS:4-6) or functional variants ofthe sequences so that the antibody or antibody fragment can bind to thetumor cell without substantially binding to normal cells. Antibodies orantibody fragments of the invention also include antibodies or antibodyfragments that bind to CD44E; alpha-fetoprotein; a protein having amolecular weight between 47-53 kDa and an isoelectric point between5.2-5.5, preferably 5.4; a protein having a molecular weight between48-54 kDa and an isoelectric point between 5.1-5.4, preferably 5.2; or aprotein comprising the amino acid sequence 107 to 487 of AFP (SEQ IDNO:14), 107 to 590 of AFP (SEQ ID NO: 15) or 107 to 609 of AFP (SEQ IDNO:16). In addition, antibodies or antibody fragments of the inventioninclude antibodies or antibody fragments that bind to a proteincomprising the 5-v8 interface of CD44E, the v8 exon of CD44 or aminoacid sequence ATNMDSSHSIT.

By “at least moderately stringent hybridization conditions” it is meantthat conditions are selected which promote selective hybridizationbetween two complementary nucleic acid molecules in solution.Hybridization may occur to all or a portion of a nucleic acid sequencemolecule. The hybridizing portion is typically at least 15 (e.g. 20, 25,30, 40 or 50) nucleotides in length. Those skilled in the art willrecognize that the stability of a nucleic acid duplex, or hybrids, isdetermined by the Tm, which in sodium containing buffers is a functionof the sodium ion concentration and temperature (Tm=81.5° C.−16.6 (Log10 [Na+])+0.41(% (G+C)−600/l), or similar equation). Accordingly, theparameters in the wash conditions that determine hybrid stability aresodium ion concentration and temperature. In order to identify moleculesthat are similar, but not identical, to a known nucleic acid molecule a1% mismatch may be assumed to result in about a 1° C. decrease in Tm,for example if nucleic acid molecules are sought that have a >95%identity, the final wash temperature will be reduced by about 5° C.Based on these considerations those skilled in the art will be able toreadily select appropriate hybridization conditions. In preferredembodiments, stringent hybridization conditions are selected. By way ofexample the following conditions may be employed to achieve stringenthybridization: hybridization at 5× sodium chloride/sodium citrate(SSC)/5×Denhardt's solution/1.0% SDS at Tm−5° C. based on the aboveequation, followed by a wash of 0.2×SSC/0.1% SDS at 60° C. Moderatelystringent hybridization conditions include a washing step in 3×SSC at42° C. It is understood, however, that equivalent stringencies may beachieved using alternative buffers, salts and temperatures. Additionalguidance regarding hybridization conditions may be found in: CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y., 1989,6.3.1-6.3.6 and in: Sambrook et al., Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press, 1989, Vol. 3.

The term “binding protein” as used herein refers to proteins thatspecifically bind to another substance. In an embodiment, bindingproteins are antibodies or antibody fragments.

The term “binding proteins of the invention” as used herein includesantibodies or antibody fragments of the invention.

By “biologically compatible form suitable for administration in vivo” ismeant a form of the substance to be administered in which any toxiceffects are outweighed by the therapeutic effects.

The term “cancer” as used herein includes any cancer that can be boundby a binding protein of the invention, preferably an antibody orantibody fragment of the invention.

The term “CD44” as used herein refers to the family of CD44 moleculesencoded by a single gene comprising a total of 19 exons. There are 10variable exons. Alternative splicing in the variable regions results inthe creation of a number of different CD44 variants (See FIG. 17). Theterm “CD44E”, also known as CD44v8-10, refers to the epithelial variantof CD44. CD44E contains variant exons 8-10 in addition to exons 1-5 and16-19. The term “v8 exon of CD44” refers to variable exon 8 of CD44. Theterm “5-v8 interface of CD44E” refers to the region where exon 5connects with variable exon 8 in CD44E. It is a continuous sequence thatincludes part of the region of exon 5 and part of the variable exon 8 ofCD44E.

A “conservative amino acid substitution”, as used herein, is one inwhich one amino acid residue is replaced with another amino acid residuewithout abolishing the protein's desired properties.

The term “controlled release system” as used means the immunoconjugateand/or other cancer therapeutic of the invention can be administered ina controlled fashion. For example, a micropump may deliver controlleddoses directly into the area of the tumor, thereby finely regulating thetiming and concentration of the pharmaceutical composition (see, e.g.,Goodson, 1984, in Medical Applications of Controlled Release, vol. 2,pp. 115-138).

The term “direct administration” as used herein means theimmunoconjugate and/or other cancer therapeutic may be administered,without limitation, intratumorally, intravascularly, and peritumorally.For example, the immunoconjugate may be administered by one or moredirect injections into the tumor, by continuous or discontinuousperfusion into the tumor, by introduction of a reservoir of theimmunoconjugate, by introduction of a slow-release apparatus into thetumor, by introduction of a slow-release formulation into the tumor,and/or by direct application onto the tumor. By the mode ofadministration “into the tumor,” introduction of the immunoconjugateand/or other cancer therapeutic to the area of the tumor, or into ablood vessel or lymphatic vessel that substantially directly flows intothe area of the tumor, is included.

As used herein, the phrase “effective amount” means an amount effective,at dosages and for periods of time necessary to achieve the desiredresult. Effective amounts of an immunoconjugate may vary according tofactors such as the disease state, age, sex, weight of the animal.Dosage regime may be adjusted to provide the optimum therapeuticresponse. For example, several divided doses may be administered dailyor the dose may be proportionally reduced as indicated by the exigenciesof the therapeutic situation.

The term “heavy chain complementarity determining region” as used hereinrefers to regions of hypervariability within the heavy chain variableregion of an antibody molecule. The heavy chain variable region hasthree complementarity determining regions termed heavy chaincomplementarity determining region 1, heavy chain complementaritydetermining region 2 and heavy chain complementarity determining region3 from the amino terminus to carboxy terminus.

The term “heavy chain variable region” as used herein refers to thevariable region of a heavy chain.

The term “immunoconjugate of the invention” is used herein comprises (1)a binding protein, preferably an antibody or antibody fragment, of theinvention attached to (2) an effector molecule. The effector moleculecan be any molecule that one wishes to deliver to the cancer cell,including, but not limited to (i) a label, which can generate adetectable signal, directly or indirectly, or (ii) a cancer therapeuticagent, such as a toxin that is either cytotoxic, cytostatic or otherwiseprevents or reduces the ability of the cancer cells to divide and/ormetastasize.

The term “isolated nucleic acid sequences” as used herein refers to anucleic acid substantially free of cellular material or culture mediumwhen produced by recombinant DNA techniques, or chemical precursors, orother chemicals when chemically synthesized. An isolated nucleic acid isalso substantially free of sequences which naturally flank the nucleicacid (i.e. sequences located at the 5′ and 3 ends of the nucleic acid)from which the nucleic acid is derived. The term “nucleic acid” isintended to include DNA and RNA and can be either double stranded orsingle stranded.

The term “isolated proteins”, such as light chain complementarityregions 1, 2 and 3, heavy chain complementarity regions 1, 2 and 3,light chain variable regions, heavy chain variable regions, and bindingproteins of the invention, refers to a protein substantially free ofcellular material or culture medium when produced by recombinant DNAtechniques, or chemical precursors or other chemicals when chemicallysynthesized.

The term “light chain complementarity determining region” as used hereinrefers to regions of hypervariability within the light chain variableregion of an antibody molecule. Light chain variable regions have threecomplementarity determining regions termed light chain complementaritydetermining region 1, light chain complementarity determining region 2and light chain complementarity determining region 3 from the aminoterminus to the carboxy terminus.

The term “light chain variable region” as used herein refers to thevariable region of a light chain.

The term “modified bouganin” as used here means a modified bouganin thathas a reduced propensity to activate an immune response as described inPCT/CA2005/000410 and U.S. patent application Ser. No. 11/084,080. Inone example, the modified bouganin has the amino acid sequence (SEQ IDNO: 17):

YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDKRFVLVDITTTSKKTVKVAIDVTDVYWGYQDKWDGKDRAVFLDKVPTVATSKLFPGVTNRVTLTFDGSYQKLVNAAKADRKALELGVNKLEFSIEAIHGKTINGQEAAKFFLIVIQMVSEAARFKYIETEWDRGLYGSFKPNFKVLNLENNWGDISDAIHKSSPQCTTINPALQLISPSNDPW WNKVSQISPDMGILKFKSSK.

The term “nucleic acid sequence” as used herein refers to a sequence ofnucleoside or nucleotide monomers consisting of naturally occurringbases, sugars and intersugar (backbone) linkages. The term also includesmodified or substituted sequences comprising non-naturally occurringmonomers or portions thereof. The nucleic acid sequences of the presentinvention may be deoxyribonucleic acid sequences (DNA) or ribonucleicacid sequences (RNA) and may include naturally occurring bases includingadenine, guanine, cytosine, thymidine and uracil. The sequences may alsocontain modified bases. Examples of such modified bases include aza anddeaza adenine, guanine, cytosine, thymidine and uracil; and xanthine andhypoxanthine.

The term “sequence identity” as used herein refers to the percentage ofsequence identity between two polypeptide sequences. In order todetermine the percentage of identity between two polypeptide sequences,the amino acid sequences of such two sequences are aligned, preferablyusing the Clustal W algorithm (Thompson, J D, Higgins D G, Gibson T J,1994, Nucleic Acids Res. 22 (22): 4673-4680), together with BLOSUM 62scoring matrix (Henikoff S, and Henikoff J. G., 1992, Proc. Natl. Acad.Sci. USA 89: 10915-10919) and a gap opening penalty of 10 and gapextension penalty of 0.1, so that the highest order match is obtainedbetween two sequences wherein at least 50% of the total length of one ofthe sequences is involved in the alignment. Other methods that may beused to align sequences are the alignment method of Needleman and Wunsch(J. Mol. Biol., 1970, 48: 443), as revised by Smith and Waterman (Adv.Appl. Math., 1981, 2: 482) so that the highest order match is obtainedbetween the two sequences and the number of identical amino acids isdetermined between the two sequences. Other methods to calculate thepercentage identity between two amino acid sequences are generally artrecognized and include, for example, those described by Carillo andLipton (SIAM J. Applied Math., 1988, 48:1073) and those described inComputational Molecular Biology, Lesk, e.d. Oxford University Press, NewYork, 1988, Biocomputing: Informatics and Genomics Projects. Generally,computer programs will be employed for such calculations. Computerprograms that may be used in this regard include, but are not limitedto, GCG (Devereux et al., Nucleic Acids Res., 1984, 12: 387) BLASTP,BLASTN and FASTA (Altschul et al., J. Molec. Biol., 1990: 215:403).

As used herein, the phrase “treating cancer” refers to inhibition ofcancer cell replication, inhibition of cancer spread (metastasis),inhibition of tumor growth, reduction of cancer cell number or tumorgrowth, decrease in the malignant grade of a cancer (e.g., increaseddifferentiation), or improved cancer-related symptoms.

The term “variant” as used herein includes modifications or chemicalequivalents of the amino acid and nucleotide sequences of the presentinvention that perform substantially the same function as the proteinsor nucleic acid molecules of the invention in substantially the sameway. For example, variants of proteins of the invention include, withoutlimitation, conservative amino acid substitutions. Variants of proteinsof the invention also include additions and deletions to the proteins ofthe invention.

The term “variant of alpha-fetoprotein” includes variants ofalpha-fetoprotein, such as a protein comprising the amino acid sequenceof SEQ ID NO:14, 15 or 16; or a protein that is a truncated version ofalpha-fetoprotein and has the molecular weight of 48-54 kDa and anisoelectric point between 5.1-5.4.

(B) Proteins and Nucleic Acids of the Invention

(i) Light and Heavy Chain Complementarity Determining Regions and Lightand Heavy Chain Variable Regions

The invention provides isolated light chain complementarity determiningregion 1 comprising the amino acid sequence SGDNLGNKYVC (SEQ ID NO:1).The invention also provides isolated light chain complementaritydetermining region 2 comprising the amino acid sequence EDTKRPS (SEQ IDNO:2). In addition, the invention provides isolated light chaincomplementarity determining region 3 comprising the amino acid sequenceQAWDSRTEI (SEQ ID NO:3).

The invention provides isolated light chain complementarity determiningregion 1 comprising the amino acid sequence GDEYYWS (SEQ ID NO:4). Theinvention also provides isolated light chain complementarity determiningregion 2 comprising the amino acid sequence YMSYRGSSYYSPSLQS (SEQ IDNO:5). In addition, the invention provides isolated light chaincomplementarity determining region 3 comprising the amino acid sequenceKYCGGDCRSGFDI (SEQ ID NO:6).

The invention provides isolated light chain complementarity determiningregions 1, 2 and 3, comprising the amino acid sequences SGDNLGNKYVC (SEQID NO:1), EDTKRPS (SEQ ID NO:2) and QAWDSRTEI (SEQ ID NO:3),respectively; and isolated heavy chain complementarity determiningregions 1, 2 and 3, comprising the amino acid sequences GDEYYWS (SEQ IDNO:4), YMSYRGSSYYSPSLQS (SEQ ID NO:5) and KYCGGDCRSGFDI (SEQ ID NO:6),respectively.

The invention also includes variants of the CDR sequences that can bindto the same epitope or antigen recognized by the CDR sequences disclosedabove.

Additional aspects of the invention are isolated light chain variableregions comprising light chain complementarity determining regions 1, 2and/or 3 of the invention (SEQ ID NOS:1-3); and heavy chain variableregions comprising the heavy chain complementarity determining regions1, 2 and/or 3 of the invention (SEQ ID NOS:4-6). In one embodiment, thelight chain variable region comprises the amino acid sequence shown inFIG. 1 (SEQ ID NO:7), and the heavy chain variable region comprises theamino acid sequence shown in FIG. 2 (SEQ ID NO:9).

The invention also includes variants of the isolated light chainvariable regions and heavy chain variable regions that can bind to thesame epitope or antigen recognized by the isolated light chain variableregions and isolated heavy chain variable regions disclosed above.

A person skilled in the art will appreciate that the invention includesvariants to the amino acid sequences of SEQ ID NOS:1-6, 7 and 9,including chemical equivalents to the sequences disclosed by the presentinvention. Such equivalents include proteins that perform substantiallythe same function as the specific proteins disclosed herein insubstantially the same way. A functional variant of a CDR sequence willbe able to bind to the antigen or epitope recognized by the native CDRsequence. For example, equivalents include, without limitation,conservative amino acid substitutions.

In one embodiment, the variant amino acid sequences of the light chaincomplementarity determining regions 1, 2 and 3, and the heavy chaincomplementarity determining regions 1, 2 and 3 have at least 50%,preferably at least 60%, more preferably at least 70%, most preferablyat least 80%, and even more preferably at least 90% sequence identity toSEQ ID NOS:1-6, respectively.

In another embodiment, the variant amino acid sequences of the lightchain variable region and the heavy chain variable region have at least50%, preferably at least 60%, more preferably at least 70%, mostpreferably at least 80%, and even more preferably at least 90% sequenceidentity to SEQ ID NOS:7 and 9, respectively.

The invention also provides an isolated nucleic acid sequence encodingthe light chain variable region of the invention, and an isolatednucleic acid sequence encoding the heavy chain variable region of theinvention. In one embodiment, the light chain variable region comprisesthe nucleic acid sequence shown in FIG. 1 (SEQ ID NO:8). In anotherembodiment, the heavy chain variable region comprises the nucleic acidsequence shown in FIG. 2 (SEQ ID NO:10). The invention also includesvariants to the nucleic acid sequences that encode for the light chainvariable region and heavy chain variable region of the invention. Forexample, the variants include nucleotide sequences that hybridize to thenucleic acid sequences encoding the light chain variable region andheavy chain variable region of the invention under at least moderatelystringent hybridization conditions.

The invention also provides isolated nucleic acid sequences encodinglight chain complementarity determining regions 1, 2 and/or 3,comprising the amino acid sequences SGDNLGNKYVC (SEQ ID NO:1), EDTKRPS(SEQ ID NO:2) and QAWDSRTEI (SEQ ID NO:3), respectively; and isolatednucleic acid sequences encoding heavy chain complementarity determiningregions 1, 2 and/or 3, comprising the amino acid sequences GDEYYWS (SEQID NO:4), YMSYRGSSYYSPSLQS (SEQ ID NO:5) and KYCGGDCRSGFDI (SEQ IDNO:6), respectively. The invention also includes isolated nucleic acidsequences encoding variants of the CDR sequences discussed above.Nucleic acid sequences encoding variants of the CDR sequences of theinvention include nucleic acid sequences that hybridize to the CDRsequences encoding the amino acid sequences shown in SEQ ID NOS:1-6under at least moderately stringent hybridization conditions.

(ii) Binding Proteins

Another aspect of the invention is a binding protein, preferably anantibody or antibody fragment, that comprises at least one light chaincomplementarity determining region of the invention (i.e. one or more ofSEQ ID NOS:1-3) and/or at least one heavy chain complementaritydetermining region of the invention (i.e. one or more of SEQ IDNOS:4-6). Such a binding protein can be generally referred to herein as“a binding protein of the invention”, or preferably “an antibody orantibody fragment of the invention”.

In one embodiment, the binding protein, preferably an antibody orantibody fragment, comprises the light chain complementarity determiningregions 1, 2 and 3, comprising the amino acid sequences SGDNLGNKYVC (SEQID NO:1), EDTKRPS (SEQ ID NO:2) and QAWDSRTEI (SEQ ID NO:3),respectively; and heavy chain complementarity determining regions 1, 2and 3, comprising the amino acid sequences GDEYYWS (SEQ ID NO:4),YMSYRGSSYYSPSLQS (SEQ ID NO:5) and KYCGGDCRSGFDI (SEQ ID NO:6),respectively. The invention also provides a binding protein, preferablyan antibody or antibody fragment, that comprises the light chainvariable region of the invention and/or the heavy chain variable regionof the invention.

A person skilled in the art will appreciate that the invention includesvariants to the specific binding proteins disclosed above, includingchemical equivalents to the sequences disclosed above that performsubstantially the same function as the binding proteins disclosed abovein substantially the same way. A functional variant of a binding proteinwill be able to bind to a protein comprising 5-v8 interface of CD44E,the v8 exon of CD44, the amino acid sequence ATNMDSSHSIT, amino acid SEQID NOS:14, 15 or 16, or to a protein having a molecular weight between47-53 kDa and an isoelectric point between 5.2-5.5; a protein having amolecular weight between 48-54 kDa and an isoelectric point between5.1-5.4, CD44E, or alpha-fetoprotein or a variant thereof.

As stated above, the inventors have identified the antigen that binds tothe binding protein of the invention. In particular, the inventors haveshown that the binding proteins of the invention bind to theextracellular domain of CD44E. In addition, the inventors have shownthat the binding proteins of the invention bind to AFP or a variantthereof.

It is important to recognize that CD44 molecules have a high potentialfor N- and O-glycosylation and for the addition of chondroitin sulfateand heparan sulfate. However, the pattern of these post-translationalmodifications is variable, and appears to be cell-specific and canpotentially affect the ability of CD44 to bind HA or other extracellularmolecules. The variable pattern of post-translational modifications isparticularly relevant to the preparation of anti-CD44 monoclonalantibodies since antibody binding has been shown to be affected by thepresence of these modifications, despite the primary structure of themolecule being the same as that of the antigen used to raise theantibody (Matzuki et al. Cancer Res 63:8278-83, 2003; Martegani et al.Amer J Pathol 154(1): 291-300, 1999). This also limits the usefulness ofrecombinant CD44 as an immunogen since its glycosylation pattern wouldlikely differ from that of tumor cells. The binding proteins of theinvention is, therefore, particularly unique since it recognizes a formof the CD44 that is present on human tumor cells.

Accordingly, the invention provides a binding protein of the inventionthat binds to a protein comprising the 5-v8 interface of CD44E, the v8exon of CD44 or amino acid sequence ATNMDSSHSIT. The invention alsoprovides a binding protein of the invention that binds to CD44E;alpha-fetoprotein; a protein having a molecular weight between 47-53 kDaand an isoelectric point between 5.2-5.5, preferably 5.4; a proteinhaving a molecular weight between 48-54 kDa and an isoelectric pointbetween 5.1-5.4, preferably 5.2; or a protein comprising the amino acidsequence 107 to 487 of AFP (SEQ ID NO:14), 107 to 590 of AFP (SEQ IDNO:15) or 107 to 609 of AFP (SEQ ID NO:16). The invention also providesa binding protein of the invention that binds to a protein comprisingSEQ ID NOS:38, 39, 40, 41, 42, 43, 44 or 45 and having a molecularweight between 47-53 kDa and an isoelectric point between 5.2-5.5; or aprotein comprising SEQ ID NOS:46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74 or 75 and having a molecular weight between 48-54 kDa and anisoelectric point between 5.1-5.4.

The invention also includes binding proteins that bind to the amino acidsequence ATNMDSSHSIT.

In certain embodiments, the antibody or antibody fragment comprises allor a portion of a heavy chain constant region, such as an IgG1, IgG2,IgG3, IgG4, IgA1, IgA2, IgE, IgM or IgD constant region. Preferably, theheavy chain constant region is an IgG1 heavy chain constant region.Furthermore, the antibody or antibody fragment can comprise all or aportion of a kappa light chain constant region or a lambda light chainconstant region. Preferably, the light chain constant region is a lambdalight chain constant region.

To produce monoclonal antibodies derived from humans, antibody producingcells (lymphocytes) can be harvested from a human having cancer andfused with myeloma cells by standard somatic cell fusion procedures thusimmortalizing these cells and yielding hybridoma cells. Such techniquesare well known in the art, (e.g. the hybridoma technique originallydeveloped by Kohler and Milstein (Nature 256:495-497 (1975)) as well asother techniques such as the human B-cell hybridoma technique (Kozbor etal., Immunol. Today 4:72 (1983)), the EBV-hybridoma technique to producehuman monoclonal antibodies (Cole et al., Methods Enzymol, 121:140-67(1986)), and screening of combinatorial antibody libraries (Huse et al.,Science 246:1275 (1989)). Another example of making human monoclonalantibodies is described in WO/9947929. In another example, amyeloma-like fusion partner, as described in Dan et al. (J Neurosurgery76:660-69, 1992) can be used. Hybridoma cells can be screenedimmunochemically for production of antibodies specifically reactive withcancer cells and the monoclonal antibodies can be isolated.

Specific antibodies, or antibody fragments, reactive against particularantigens or molecules, such as antigens or molecules on a cancer cell,may also be generated by screening expression libraries encodingimmunoglobulin genes, or portions thereof, expressed in bacteria withcell surface components. For example, complete Fab fragments, VH regionsand FV regions can be expressed in bacteria using phage expressionlibraries (See for example Ward et al., Nature 34J.:544-546 (1989); Huseet al., Science 246:1275-1281 (1989); and McCafferty et al., Nature348:552-554 (1990)). The present invention includes all antibodies andantibody fragments that bind to the same antigen as the antibodies orantibody fragments of the invention. A person skilled in the art willappreciate that binding assays can be used to find other antibodies andantibody fragments with the same binding specificities as the antibodiesand antibody fragments of the invention. As exemplified, below, acompetition binding assay can be used to find such other antibodies.

Before a competition assay is performed using flow cytometry, theminimal concentration of antibody of the invention (Ab1) that givesmaximal binding against a fixed number of tumor cells (for example,A-375 cells for VB1-008) is determined. A total of 10⁶ cells areharvested from exponentially growing cultures and incubated with variousantibody concentrations for 1 hr at 4° C. The cells are washed andincubated with a suitable detection antibody for an additional hour at4° C. After washing, the cells are analyzed by flow cytometry. For eachtest antibody, a saturation curve is generated from the data by plottingmedian fluorescence against the antibody concentration.

For the competition assay, tumor cells are prepared as above and treatedin duplicate with a fixed concentration of antibody (Ab1). The fixedconcentration is the minimal concentration of antibody that generatesmaximal binding against a fixed number of tumor cells as determinedabove. Immediately following the addition of the Ab1, varyingconcentrations of the potential inhibitory antibody (Ab2) is added toeach tube and the mixture incubated for 1 hr at 4° C. Both the percentinhibition and change over maximum median fluorescence are calculated bysubtracting the background fluorescence (PBS-5% FCS) from the medianfluorescence reading for each test sample (Ab1+Ab2). The result is thendivided by the median fluorescence of Ab1 alone (maximal binding) minusthe background (see below). The percent of inhibition result is obtainedby multiplying by 100. The mean of the replicates along with theirrespective standard error is plotted against antibody concentration. Thefollowing formula is used to calculate the percent inhibition:PI=[MF(_(Ab1+Ab2))−MF_(Bgd))/(MF_(Ab1)−MF_(Bgd))]×100

where PI=percent inhibition; MF(_(Ab1+Ab2))=median fluorescence measuredfor Ab1+Ab2 mixture; and MF_(Bgd)=background median fluorescence withPBS-5% FCS.

Accordingly, the invention provides a binding protein capable of bindingan antigen on a tumor cell wherein the binding protein can be identifiedby a method comprising:

-   -   (1) incubating a fixed number of tumor cells with a minimal        concentration of a binding protein of the invention, preferably        an antibody or antibody fragment

(Ab1) that generates maximal binding against the fixed number of tumorcells and measuring median fluorescence of Ab1 (MF_(Ab1));

-   -   (2) testing two or more concentrations of a test binding protein        (Ab2) by adding Ab2 to the Ab1 and tumor cells, and measuring        median fluorescence (MF_((Ab1+Ab2)));    -   (3) measuring background median fluorescence (MF_(bgd));    -   (4) calculating PI, wherein        PI=[MF(_(Ab1+Ab2))−MF_(Bgd))/(MF_(Ab1)−MF_(Bgd))]×100; and    -   (5) comparing the PI to a control PI value;

wherein, a PI that has a statistically significant difference from thecontrol PI indicates that the test binding protein is capable of bindingthe antigen on the tumor cell.

The competition binding assay can also be done with peptides, preferablythe peptide defined by SEQ ID NO:28. Similar to the method describedabove, before the competition assay is performed, the minimalconcentration of test binding protein (Ab2) that gives maximal bindingagainst a fixed number of tumor cells is determined.

Accordingly, an embodiment of the invention provides a binding proteincapable of binding an antigen on a tumor cell wherein the bindingprotein can be identified by a method comprising:

-   -   (1) incubating a fixed number of tumor cells with a minimal        concentration of a test binding protein (Ab2) that generates        maximal binding against the fixed number of tumor cells and        measuring median fluorescence of Ab2 (MF_(Ab2));    -   (2) preparing a peptide and Ab2 mixture by incubating a molar        excess of a peptide defined by SEQ ID NO:28 with said minimal        concentration of the test binding protein (Ab2);    -   (3) adding said mixture to tumor cells and measuring median        fluorescence (MF_((Ab2+peptide)));    -   (4) measuring background median fluorescence (MF_(bgd));    -   (5) calculating PI, wherein        PI=[MF(_(Ab2+Peptide))−MF_(Bgd))/(MF_(Ab2)−MF_(Bgd))]×100; and    -   (6) comparing the PI to a control PI value;

wherein, a PI that has a statistically significant difference from thecontrol PI indicates that the test binding protein is capable of bindingthe antigen on the tumor cell.

A person skilled in the art will appreciate that affinity maturationtechniques could be used modify the binding proteins or immunoconjugatesof the invention either by increasing its affinity for both CD44E andAFP or by selecting out the binding to one antigen. The latter can leadto the development of 2 separate antibodies or immunoconjugates withpreferential binding to either AFP or to CD44E.

Two strategies are routinely used to enhance the binding affinity of anantibody. One approach utilizes the resolution of the crystal structureof the Ab-Ag complex to identify the key residues involved in theantigen binding (Davies D. R., Cohen G. H. 1996. Interactions of proteinantigens with antibodies. Proc Natl. Acad. Sci. U.S.A. 93, 7-12).Subsequently, those residues can be mutated to enhance the interaction.The other approach mimics an in vivo antigen stimulation that drives theaffinity maturation of immunoglobulin produced by B cells. During thematuration of the immune response, the variable regions of theimmunoglobulins are subjected to somatic mutations (Me Heyzer-WilliamsM. 2003. B-cell signaling mechanism and activation. FundamentalImmunology, Fifth edition, 195-225). This process, highly specific forthe immune system, is characterized by the introduction of pointmutations at a very high rate. It occurs only within the DNA fragmentsencoding the variable regions and excludes the conserved domains. The Bcells expressing the somatically mutated variant antibody are thensubjected to an antigen-mediated selection resulting in the selection ofhigher affinity immunoglobulin. In order to replicate this phenomenon invitro, several approaches have been used to introduce mutations eitherby random or targeted processes. The random mutations can be introducedusing error-prone PCR, chain shuffling or mutator E. coli strains(Clackson T. Hoogenboom N. R., Griffiths A. D. and Winter G. 1991 Makingantibody fragments using phage display libraries. Nature 352, 624-628,Hawkins R. E., Russell S. J. and Winter G. 1992. Selection of phageantibodies by binding affinity. Mimicking affinity maturation. J. Mol.Biol. 226, 889-896, Low N., Holliger P. and Winter G. 1996. Mimickingsomatic hypermutation: affinity maturation of antibodies displayed onbacteriophage using a bacterial mutator strain. J. Mol. Biol. 260,359-368). This strategy leads to the creation of large libraries inwhich reactive clones are selected with a display technology such asribosome, phage or yeast (Min L. (2000). Applications of displaytechnology in protein analysis. Nat. Biotechnol. 18, 1251-1256).

The targeted mutations of the CDRs, especially CDR3 of the light andheavy chains, have been shown to be an effective technique forincreasing antibody affinity. Blocks of 3 to 4 amino acids of the CDR3or specific regions called “hot-spots” are targeted for mutagenesis.Yang et al reported an increase of 420 fold of an anti-HIV gp120 Fabfragment by mutating four CDR residues (Yang W. P., Green K.,Pinz-Sweeney S., Briones A T., Burton D. R. and Barbas C. F. III. 1995.CDR walking mutagenesis for the affinity maturation of a potent humananti-HIV-1 antibody into picomolar range. J. Mol. Biol., 254, 392-403).One mutation in the VL CDR3 combined with three mutations in the VH CDR3of the C6.5 scFv yielded a 1230 fold increased affinity (Schier R.,McCall A., Adams G. P., Marshall K. W., Merrit H., Yin M., Crawford R.S. Weiner L. M., Marks C. and Marks J. D. 1996. Isolation of picomolaraffinity anti-c-erbB-2 single-chain Fv by molecular evolution of thecomplementary determining regions in the center of the antibody bindingsite. J. Mol. Biol, 263, 551-567).

“Hot spots” are the sequences where somatic hypermutation takes place invivo (Neuberger M. S and Milstein C. 1995. Somatic hypermutation. Curr.Opin. Immunol. 7, 248-254). The hotspot sequences can be defined asconsensus nucleotide sequences in certain codons. The consensus sequenceis the tetranucleotide, RGYW, in which R can be either A or G, Y can beC or T and W can be either A or T (Neuberger M. S and Milstein C. 1995.Somatic hypermutation. Curr. Opin. Immunol. 7, 248-254). In addition,the serine residues encoded by the nucleotides AGY are predominantlypresent in the CDRs regions of the variable domain over those encoded byTCN corresponding to a potential hot-spot sequences (Wagner S. D.,Milstein C. and Neuberger M. S. 1995. Codon bias targets mutation.Nature, 376, 732). The structural analysis has shown that the CDR loopscontribute the most to the antigen binding, especially the CDR3 loops(Giudicelli V., Chaume D. and Lefranc M. P. 2004. IMGTA/-QUEST, anintegrated software program for immunoglobulin and T cell receptor V-Jand V-D-J rearrangement analysis. Nucleis Acids Res. 32, 435-440).Therefore, the nucleotide sequence of the CDRs of the heavy and lightchains of each antibody of the invention is scanned for the presence ofthe hot-spot sequences and AGY codons. The identified hot-spots of theCDR regions of the light and heavy chain are compared to the germinalsequences of the heavy and light chains using the InternationalImMunoGen Tics database (IMGT, imgt.cines.fr/textes/vquest/) (Davies D.R., Padlan E. A. and Sheriff S. 1990. Antibody-antigen complexes. Annu.Rev. Biochem. 59, 439-473). A sequence, identical to the germ line,suggest that somatic mutation has not occurred; therefore the randommutations are introduced mimicking the somatic events occurring in vivo.In contrast, a different sequence shows that some somatic mutations havealready occurred. It will remain to be determined if the in vivo somaticmutation was optimal. The hot-spots that code for buried or conservedamino acids within the CDRs are not mutagenized. These residues areusually critical for the overall structure and are unlikely to interactwith the antigen since they are buried. In addition, the sequences canbe compared to the predicted locations in the germ line sequences wheresomatic mutations occurred predominantly (Tomlinson I. M., Cox J. P. L.,Gherardi E., Lesk A. M. and Chotia C. 1995. The structural repertoire ofthe human Vldomain. EMBO J. 14, 4628-4638, Tomlinson I. M., Walter G.,Jones P. T., Dear P. H., Sonnhammer E. L. L and Winter G. 1996. Theimprint of somatic hypermutation on the repertoire of human germline Vgenes. J. Mol. Biol. 256, 813-817). A similar strategy was applied forthe affinity maturation of BL22 scFv. A point mutation introduced in theCDR3 of the heavy resulted in 5 to 10 fold increase in binding activityon various CD22-positive cell lines (Salvatore G., Beers R., MarguliesI., Kreitman R. J. and Pastan I. 2002. Improved cytotoxic activitytoward cell lines and fresh leukemia cells of a mutant anti-CD22immunotoxin obtained by antibody phage display. Clinical Cancerresearch, 8, 995-1002). Also, the mutation of various amino acids in theCDR1 and CDR2 loops also produced mutant with increase affinity rangingfrom 3 fold to 7 fold (Ho M., Kreitman J., Onda M. and Pastan I. 2005.In vitro antibody evolution targeting germline hot spots to increaseactivity of an anti-CD22 immunotoxin. J. Biol. Chem., 280, 607-617).

After mutations are introduced, either by random or targeted processes,the antibodies are expressed and assessed for function. For instance,functional screening can be based on binding. Once function is assessed,then DNA sequencing of the chosen antibodies can be carried out usingknown methods.

In another embodiment, the anchored periplasmic expression (APEx) methoddescribed by Harvey, B et al (PNAS 2004 Jun. 22; 101(25): 9193-8) isused for affinity maturation of the binding proteins or immunoconjugatesof the invention.

Accordingly, the invention includes binding proteins of the inventionthat have been affinity maturized to increase the affinity of thebinding protein to CD44E and AFP or a variant thereof, or to select abinding protein that has affinity to CD44E or AFP or a variant thereof.

The invention also provides compositions comprising the binding proteinsof the invention, preferably antibodies and antibody fragments, with apharmaceutically acceptable excipient, carrier, buffer or stabilizer.

(C) Immunoconjugates

The invention also includes an immunoconjugate comprising (1) a bindingprotein of the invention, preferably an antibody or antibody fragment,that has been attached to (2) an effector molecule. In one embodiment,the binding protein of the invention binds to an antigen or molecule onor in a cancer cell.

The antigen can be a protein comprising the 5-v8 interface of CD44E; aprotein comprising the v8 exon of CD44; CD44E; a protein comprisingamino acid sequence ATNMDSSHSIT; alpha-fetoprotein or a variant thereof;a protein having a molecular weight between 47-53 kDa and an isoelectricpoint between 5.2-5.5, preferably 5.4; a protein having a molecularweight between 48-54 kDa and an isoelectric point between 5.1-5.4,preferably 5.2; or a protein comprising the amino acid sequence 107 to487 of AFP (SEQ ID NO:14), 107 to 590 of AFP (SEQ ID NO:15) or 107 to609 of AFP (SEQ ID NO:16). In another example the antigen is a proteincomprising amino acid SEQ ID NOS: 38, 39, 40, 41, 42, 43, 44 or 45 andhaving a molecular weight between 47-53 kDa and an isoelectric pointbetween 5.2-5.5; or a protein comprising amino acid SEQ ID NOS: 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74 or 75 and having a molecular weightbetween 48-54 kDa and an isoelectric point between 5.1-5.4.

In a preferred, embodiment the effector molecule is (i) a label, whichcan generate a detectable signal, directly or indirect, or (ii) a cancertherapeutic agent, which is either cytotoxic, cytostatic or otherwiseprevents or reduces the ability of the cancer cells to divide and/ormetastasize. Such an immunoconjugate can be generally referred to as“the immunoconjugate of the invention” herein.

In the embodiment of the invention the effector molecule is a cancertherapeutic agent. The cancer therapeutic agent is preferably a toxinthat is either cytotoxic, cytostatic or otherwise prevents or reducesthe ability of the cancer cells to divide and/or metastasize.Accordingly, one aspect of the invention is an immunoconjugatecomprising (1) a binding protein of the invention, preferably anantibody or antibody fragment, attached to (2) a cancer therapeuticagent, such as a toxin.

In another embodiment, the immunoconjugate is internalized and thecancer therapeutic agent is a toxin that blocks the protein synthesis ofthe cell, therein leading to cell death. Importantly, since most normalcells do not widely express the antigen present on the cancer cells,they cannot bind and internalize the immunoconjugate, and are protectedfrom the killing effect of the toxin or other cancer therapeutic agents.

A variety of effector molecules may be used in the immunoconjugates ofthe invention and a number of such effector molecules areintracellularly active molecules. Accordingly, in an embodiment of theinvention, the immunoconjugate is internalized by the cancer cell.

In preferred embodiments, the effector molecule is a cancer therapeuticagent, more preferably a toxin that comprises a polypeptide havingribosome-inactivating activity including, without limitation, gelonin,bouganin, saporin, ricin, ricin A chain, bryodin, diphtheria toxin,restrictocin, Pseudomonas exotoxin A and variants thereof. When theprotein is a ribosome-inactivating protein, the immunoconjugate must beinternalized upon binding to the cancer cell in order for the toxin tobe cytotoxic to the cells. Accordingly, in an embodiment of theinvention, the effector molecule is a toxin and the immunoconjugate isinternalized by the cancer cell.

In one embodiment of the invention, the toxin is bouganin or Pseudomonasexotoxin A, and variants thereof. In another embodiment, the toxin ismodified bouganin or a truncated form of Pseudomonas exotoxin A thatconsists of amino acids 252-608.

The invention includes an immunoconjugate comprising a protein encodedby nucleic acid sequence of SEQ ID NO:11 (FIG. 20). The invention alsoincludes an immunoconjugate comprising the amino acid sequences of SEQID NO: 12 and 13 (FIG. 21).

In other nonlimiting embodiments, the cancer therapeutic agent comprisesan agent that acts to disrupt DNA. Thus, the cancer therapeutic agentsmay be selected, without limitation, from enediynes (e.g., calicheamicinand esperamicin) and non-enediyne small molecule agents (e.g.,bleomycin, methidiumpropyl-EDTA-Fe(II)). Other cancer therapeutic agentsuseful in accordance with the invention include, without limitation,daunorubicin, doxorubicin, distamycin A, cisplatin, mitomycin C,ecteinascidins, duocarmycin/CC-1065, and bleomycin/pepleomycin.

In other nonlimiting embodiments, the cancer therapeutic agent comprisesan agent that acts to disrupt tubulin. Such agents may comprise, withoutlimitation, rhizoxin/maytansine, paclitaxel, vincristine andvinblastine, colchicine, auristatin dolastatin 10 MMAE, and pelorusideA.

In other nonlimiting embodiments, the cancer therapeutic portion of animmunoconjugate of the invention may comprise an alkylating agentincluding, without limitation, Asaley NSC 167780, AZQ NSC 182986, BCNUNSC 409962, Busulfan NSC 750, carboxyphthalatoplatinum NSC 271674, CBDCANSC 241240, CCNU NSC 79037, CHIP NSC 256927, chlorambucil NSC 3088,chlorozotocin NSC 178248, cis-platinum NSC 119875, clomesone NSC 338947,cyanomorpholinodoxorubicin NSC 357704, cyclodisone NSC 348948,dianhydrogalactitol NSC 132313, fluorodopan NSC 73754, hepsulfam NSC329680, hycanthone NSC 142982, melphalan NSC 8806, methyl CCNU NSC95441, mitomycin C NSC 26980, mitozolamide NSC 353451, nitrogen mustardNSC 762, PCNU NSC 95466, piperazine NSC 344007, piperazinedione NSC135758, pipobroman NSC 25154, porfiromycin NSC 56410, spirohydantoinmustard NSC 172112, teroxirone NSC 296934, tetraplatin NSC 363812,thiotepa NSC 6396, triethylenemelamine NSC 9706, uracil nitrogen mustardNSC 34462, and Yoshi-864 NSC 102627.

In other nonlimiting embodiments, the cancer therapeutic agent portionof the immunoconjugate of the invention may comprise an antimitoticagent including, without limitation, allocolchicine NSC 406042,Halichondrin B NSC 609395, colchicine NSC 757, colchicine derivative NSC33410, dolastatin 10 NSC 376128 (NG—auristatin derived), maytansine NSC153858, rhizoxin NSC 332598, taxol NSC 125973, taxol derivative NSC608832, thiocolchicine NSC 361792, trityl cysteine NSC 83265,vinblastine sulfate NSC 49842, and vincristine sulfate NSC 67574.

In other nonlimiting embodiments, the cancer therapeutic agent portionof the immunoconjugate of the invention may comprise an topoisomerase Iinhibitor including, without limitation, camptothecin NSC 94600,camptothecin, Na salt NSC 100880, aminocamptothecin NSC 603071,camptothecin derivative NSC 95382, camptothecin derivative NSC 107124,camptothecin derivative NSC 643833, camptothecin derivative NSC 629971,camptothecin derivative NSC 295500, camptothecin derivative NSC 249910,camptothecin derivative NSC 606985, camptothecin derivative NSC 374028,camptothecin derivative NSC 176323, camptothecin derivative NSC 295501,camptothecin derivative NSC 606172, camptothecin derivative NSC 606173,camptothecin derivative NSC 610458, camptothecin derivative NSC 618939,camptothecin derivative NSC 610457, camptothecin derivative NSC 610459,camptothecin derivative NSC 606499, camptothecin derivative NSC 610456,camptothecin derivative NSC 364830, camptothecin derivative NSC 606497,and morpholinodoxorubicin NSC 354646.

In other nonlimiting embodiments, cancer therapeutic agent portion ofthe immunoconjugate of the invention may comprise an topoisomerase IIinhibitor including, without limitation, doxorubicin NSC 123127,amonafide NSC 308847, m-AMSA NSC 249992, anthrapyrazole derivative NSC355644, pyrazoloacridine NSC 366140, bisantrene HCL NSC 337766,daunorubicin NSC 82151, deoxydoxorubicin NSC 267469, mitoxantrone NSC301739, menogaril NSC 269148, N,N-dibenzyl daunomycin NSC 268242,oxanthrazole NSC 349174, rubidazone NSC 164011, VM-26 NSC 122819, andVP-16 NSC 141540.

In other nonlimiting embodiments, the cancer therapeutic agent portionof the immunoconjugate of the invention may comprise an RNA or DNAantimetabolite including, without limitation, L-alanosine NSC 153353,5-azacytidine NSC 102816, 5-fluorouracil NSC 19893, acivicin NSC 163501,aminopterin derivative NSC 132483, aminopterin derivative NSC 184692,aminopterin derivative NSC 134033, an antifol NSC 633713, an antifol NSC623017, Baker's soluble antifol NSC 139105, dichlorallyl lawsone NSC126771, brequinar NSC 368390, ftorafur (pro-drug) NSC 148958,5,6-dihydro-5-azacytidine NSC 264880, methotrexate NSC 740, methotrexatederivative NSC 174121, N-(phosphonoacetyl)-L-aspartate (PALA) NSC224131, pyrazofurin NSC 143095, trimetrexate NSC 352122, 3-HP NSC 95678,2′-deoxy-5-fluorouridine NSC 27640, 5-HP NSC 107392, alpha-TGDR NSC71851, aphidicolin glycinate NSC 303812, ara-C NSC 63878,5-aza-2′-deoxycytidine NSC 127716, beta-TGDR NSC 71261, cyclocytidineNSC 145668, guanazole NSC 1895, hydroxyurea NSC 32065, inosineglycodialdehyde NSC 118994, macbecin II NSC 330500, pyrazoloimidazoleNSC 51143, thioguanine NSC 752, and thiopurine NSC 755.

The present invention further provides immunoconjugates comprising (i) abinding protein of the invention, preferably an antibody or antibodyfragment, attached to (2) an effector molecule, wherein the effectormolecule is a label, which can generate a detectable signal, indirectlyor directly. These immunoconjugates can be used for research ordiagnostic applications, such as for the in vivo detection of cancer.The label is preferably capable of producing, either directly orindirectly, a detectable signal. For example, the label may beradio-opaque or a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, ¹²³I, ¹²⁵I,¹³¹I; a fluorescent (fluorophore) or chemiluminescent (chromophore)compound, such as fluorescein isothiocyanate, rhodamine or luciferin; anenzyme, such as alkaline phosphatase, beta-galactosidase or horseradishperoxidase; an imaging agent; or a metal ion.

In another embodiment, the immunoconjugate is detectable indirectly. Forexample, a secondary antibody that is specific for the immunoconjugateand contains a detectable label can be used to detect theimmunoconjugate.

The binding protein of the invention, preferably an antibody of antibodyfragment, may be “attached to” the effector molecule by any means bywhich the binding protein can be associated with, or linked to, theeffector molecule. For example, the binding protein may be attached tothe effector molecule by chemical or recombinant means. Chemical meansfor preparing fusions or conjugates are known in the art and can be usedto prepare the immunoconjugate. The method used to conjugate the bindingprotein and effector molecule must be capable of joining the bindingprotein with the effector molecule without interfering with the abilityof the binding protein to bind to the antigen on the cancer cell.

In one embodiment, the binding protein, preferably an antibody orantibody fragment, and effector molecule are both proteins and can beconjugated using techniques well known in the art. There are severalhundred crosslinkers available that can conjugate two proteins. (See forexample “Chemistry of Protein Conjugation and Crosslinking”. 1991, ShansWong, CRC Press, Ann Arbor). The crosslinker is generally chosen basedon the reactive functional groups available or inserted on the bindingprotein, preferably an antibody or antibody fragment, and/or effectormolecule. In addition, if there are no reactive groups, aphotoactivatible crosslinker can be used. In certain instances, it maybe desirable to include a spacer between the binding protein, preferablyan antibody or antibody fragment, and effector molecule. Crosslinkingagents known to the art include the homobifunctional agents:glutaraldehyde, dimethyladipimidate and Bis(diazobenzidine) and theheterobifunctional agents: m Maleimidobenzoyl-N-Hydroxysuccinimide andSulfo-m Maleimidobenzoyl-N-Hydroxysuccinimide.

A binding protein-effector molecule protein fusion may also be preparedusing recombinant DNA techniques. In such a case a DNA sequence encodingthe binding protein is fused to a DNA sequence encoding the effectormolecule, resulting in a chimeric DNA molecule. The chimeric DNAsequence is transfected into a host cell that expresses the fusionprotein. The fusion protein can be recovered from the cell culture andpurified using techniques known in the art.

Examples of attaching an effector molecule, which is a label, to thebinding protein include the methods described in Hunter, et al., Nature144:945 (1962); David, et al., Biochemistry 13:1014 (1974); Pain, etal., J. Immunol. Meth. 40:219 (1981); Nygren, J. Histochem. andCytochem. 30:407 (1982); Wensel and Meares, Radioimmunoimaging AndRadioimmunotherapy, Elsevier, N.Y. (1983); and Colcher et al., “Use OfMonoclonal Antibodies As Radiopharmaceuticals For The Localization OfHuman Carcinoma Xenografts In Athymic Mice”, Meth. Enzymol., 121:802-16(1986).

(D) Preparation of Proteins of the Invention

A person skilled in the art will appreciate that the proteins of theinvention, such as the light and heavy complementarity determiningregions, the light and heavy chain variable regions, antibodies andantibody fragments, and immunoconjugates, may be prepared in any ofseveral ways, but is most preferably prepared using recombinant methods.

Accordingly, the nucleic acid molecules of the present invention may beincorporated in a known manner into an appropriate expression vectorwhich ensures good expression of the proteins of the invention. Possibleexpression vectors include but are not limited to cosmids, plasmids, ormodified viruses (e.g. replication defective retroviruses, adenovirusesand adeno-associated viruses), so long as the vector is compatible withthe host cell used. The expression vectors are “suitable fortransformation of a host cell”, which means that the expression vectorscontain a nucleic acid molecule of the invention and regulatorysequences selected on the basis of the host cells to be used forexpression, which is operatively linked to the nucleic acid molecule.Operatively linked is intended to mean that the nucleic acid is linkedto regulatory sequences in a manner which allows expression of thenucleic acid.

The invention therefore contemplates a recombinant expression vector ofthe invention containing a nucleic acid molecule of the invention, or afragment thereof, and the necessary regulatory sequences for thetranscription and translation of the inserted protein-sequence.

Suitable regulatory sequences may be derived from a variety of sources,including bacterial, fungal, viral, mammalian, or insect genes (Forexample, see the regulatory sequences described in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990)). Selection of appropriate regulatory sequences isdependent on the host cell chosen as discussed below, and may be readilyaccomplished by one of ordinary skill in the art. Examples of suchregulatory sequences include: a transcriptional promoter and enhancer orRNA polymerase binding sequence, a ribosomal binding sequence, includinga translation initiation signal. Additionally, depending on the hostcell chosen and the vector employed, other sequences, such as an originof replication, additional DNA restriction sites, enhancers, andsequences conferring inducibility of transcription may be incorporatedinto the expression vector.

The recombinant expression vectors of the invention may also contain aselectable marker gene which facilitates the selection of host cellstransformed or transfected with a recombinant molecule of the invention.Examples of selectable marker genes are genes encoding a protein such asG418 and hygromycin which confer resistance to certain drugs,β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase,or an immunoglobulin or portion thereof such as the Fc portion of animmunoglobulin preferably IgG. Transcription of the selectable markergene is monitored by changes in the concentration of the selectablemarker protein such as |3-galactosidase, chloramphenicolacetyltransferase, or firefly luciferase. If the selectable marker geneencodes a protein conferring antibiotic resistance such as neomycinresistance transformant cells can be selected with G418. Cells that haveincorporated the selectable marker gene will survive, while the othercells die. This makes it possible to visualize and assay for expressionof recombinant expression vectors of the invention and in particular todetermine the effect of a mutation on expression and phenotype. It willbe appreciated that selectable markers can be introduced on a separatevector from the nucleic acid of interest.

The recombinant expression vectors may also contain genes which encode afusion moiety which provides increased expression of the recombinantprotein; increased solubility of the recombinant protein; and aid in thepurification of the target recombinant protein by acting as a ligand inaffinity purification. For example, a proteolytic cleavage site may beadded to the target recombinant protein to allow separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Typical fusion expression vectors include pGEX(Amrad Corp., Melbourne, Australia), pMal (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the recombinant protein.

Recombinant expression vectors can be introduced into host cells toproduce a transformed host cell. The terms “transformed with”,“transfected with”, “transformation” and “transfection” are intended toencompass introduction of nucleic acid (e.g. a vector) into a cell byone of many possible techniques known in the art. The term “transformedhost cell” as used herein is intended to also include cells capable ofglycosylation that have been transformed with a recombinant expressionvector of the invention. Prokaryotic cells can be transformed withnucleic acid by, for example, electroporation or calcium-chloridemediated transformation. For example, nucleic acid can be introducedinto mammalian cells via conventional techniques such as calciumphosphate or calcium chloride co-precipitation, DEAE-dextran mediatedtransfection, lipofectin, electroporation or microinjection. Suitablemethods for transforming and transfecting host cells can be found inSambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition,Cold Spring Harbor Laboratory press (1989)), and other laboratorytextbooks.

Suitable host cells include a wide variety of eukaryotic host cells andprokaryotic cells. For example, the proteins of the invention may beexpressed in yeast cells or mammalian cells. Other suitable host cellscan be found in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1991). In addition,the proteins of the invention may be expressed in prokaryotic cells,such as Escherichia coli (Zhang et al., Science 303(5656): 371-3(2004)).

Yeast and fungi host cells suitable for carrying out the presentinvention include, but are not limited to Saccharomyces cerevisiae, thegenera Pichia or Kluyveromyces and various species of the genusAspergillus. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSed (Baldari. et al., Embo J. 6:229-234 (1987)), pMFa(Kurjan and Herskowitz, Cell 30:933-943 (1982)), pJRY88 (Schultz et al.,Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego,Calif.). Protocols for the transformation of yeast and fungi are wellknown to those of ordinary skill in the art (see Hinnen et al., Proc.Natl. Acad. Sci. USA 75:1929 (1978); Itoh et al., J. Bacteriology153:163 (1983), and Cullen et al. (Bio/Technology 5:369 (1987)).

Mammalian cells suitable for carrying out the present invention include,among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g. ATCC No.CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCCNo. 1573) and NS-1 cells. Suitable expression vectors for directingexpression in mammalian cells generally include a promoter (e.g.,derived from viral material such as polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40), as well as other transcriptionaland translational control sequences. Examples of mammalian expressionvectors include pCDM8 (Seed, B., Nature 329:840 (1987)) and pMT2PC(Kaufman et al., EMBO J. 6:187-195 (1987)).

Given the teachings provided herein, promoters, terminators, and methodsfor introducing expression vectors of an appropriate type into plant,avian, and insect cells may also be readily accomplished. For example,within one embodiment, the proteins of the invention may be expressedfrom plant cells (see Sinkar et al., J. Biosci (Bangalore) 11:47-58(1987), which reviews the use of Agrobacterium rhizogenes vectors; seealso Zambryski et al., Genetic Engineering, Principles and Methods,Hollaender and Setlow (eds.), Vol. VI, pp. 253-278, Plenum Press, NewYork (1984), which describes the use of expression vectors for plantcells, including, among others, PAPS2022, PAPS2023, and PAPS2034)

Insect cells suitable for carrying out the present invention includecells and cell lines from Bombyx, Trichoplusia or Spodotera species.Baculovirus vectors available for expression of proteins in culturedinsect cells (SF 9 cells) include the pAc series (Smith et al., Mol.Cell. Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow, V. A., andSummers, M. D., Virology 170:31-39 (1989)). Some baculovirus-insect cellexpression systems suitable for expression of the recombinant proteinsof the invention are described in PCT/US/02442.

Alternatively, the proteins of the invention may also be expressed innon-human transgenic animals such as, rats, rabbits, sheep and pigs(Hammer et al. Nature 315:680-683 (1985); Palmiter et al. Science222:809-814 (1983); Brinster et al. Proc. Natl. Acad. Sci. USA82:4438-4442 (1985); Palmiter and Brinster Cell 41:343-345 (1985) andU.S. Pat. No. 4,736,866).

The proteins of the invention may also be prepared by chemical synthesisusing techniques well known in the chemistry of proteins such as solidphase synthesis (Merrifield, J. Am. Chem. Assoc. 85:2149-2154 (1964);Frische et al., J. Pept. Sci. 2(4): 212-22 (1996)) or synthesis inhomogenous solution (Houbenweyl, Methods of Organic Chemistry, ed. E.Wansch, Vol. 15 I and II, Thieme, Stuttgart (1987)).

N-terminal or C-terminal fusion proteins comprising the proteins of theinvention conjugated with other molecules, such as proteins may beprepared by fusing, through recombinant techniques. The resultant fusionproteins contain a protein of the invention fused to the selectedprotein or marker protein as described herein. The recombinant proteinof the invention may also be conjugated to other proteins by knowntechniques. For example, the proteins may be coupled usingheterobifunctional thiol-containing linkers as described in WO 90/10457,N-succinimidyl-3-(2-pyridyldithio-proprionate) or N-succinimidyl-5thioacetate. Examples of proteins which may be used to prepare fusionproteins or conjugates include cell binding proteins such asimmunoglobulins, hormones, growth factors, lectins, insulin, low densitylipoprotein, glucagon, endorphins, transferrin, bombesin,asialoglycoprotein glutathione-S-transferase (GST), hemagglutinin (HA),and truncated myc.

Accordingly, the invention provides a recombinant expression vectorcomprising the nucleic acid sequences that encode the proteins of theinvention, such as the light and heavy chain complementarity determiningregions, the light and heavy chain variable regions, the bindingproteins, such as antibodies and antibody fragments, andimmunoconjugates of the invention. Further, the invention provides ahost cell comprising the recombinant expression vector of the invention.

(E) Therapeutic Methods and Pharmaceutical Compositions

The inventors have shown that binding proteins of the invention bind tothe extracellular domain of CD44E and that binding proteins of theinvention are internalized. Thus, the binding proteins of invention canbe used for the targeted delivery of bioactive or medically relevantagents, such as imaging, radioactive or cytotoxic agents.

The inventors have also shown that the binding proteins of the inventionbind to AFP or a variant thereof. Full length AFP can be found in freeform in circulation and it is internalized upon binding to its receptor.Targeting circulating AFP with the binding proteins of the invention canthus also be used for targeted drug delivery.

In one embodiment, the invention provides a method of treating orpreventing cancer, comprising administering to a patient suspected ofhaving cancer an effective amount of the immunoconjugate of theinvention, wherein the effector molecule is a cancer therapeutic agent.In another embodiment, the invention provides the use of an effectiveamount of the immunoconjugate of the invention, wherein the effectormolecule is a cancer therapeutic agent, for the manufacture of amedicament for treating or preventing cancer. Furthermore, the inventionprovides the use of an effective amount of the immunoconjugate of theinvention, wherein the effector molecule is a cancer therapeutic agent,comprising the use of an additional cancer therapeutic for themanufacture of a medicament for simultaneous, separate or sequentialtreatment or prevention of cancer.

In one embodiment of the invention, cancer includes, without limitation,cervical cancer, uterine cancer, ovarian cancer, pancreatic cancer,kidney cancer, gallbladder cancer, liver cancer, head and neck cancer,squamous cell carcinoma, gastrointestinal cancer, breast cancer (such ascarcinoma, ductal, lobular, and nipple), prostate cancer, testicularcancer, lung cancer, non-small cell lung cancer, non-Hodgkin's lymphoma,multiple myeloma, leukemia (such as acute lymphocytic leukemia, chroniclymphocytic leukemia, acute myelogenous leukemia, and chronicmyelogenous leukemia), brain cancer, neuroblastoma, sarcomas, coloncancer, rectum cancer, stomach cancer, bladder cancer, pancreaticcancer, endometrial cancer, plasmacytoma, lymphoma, and melanoma. In apreferred embodiment, the cancer includes, without limitation, bladdercancer, breast cancer, cervical cancer, colon cancer, kidney cancer,liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostatecancer, rectal cancer, skin cancer, stomach cancer, uterine cancer, andhead and neck cancer.

The ability of the immunoconjugate of the invention to selectivelyinhibit or destroy cancerous cells may be readily tested in vitro usingcancer cell lines. The selective inhibitory effect of theimmunoconjugates of the invention may be determined, for example bydemonstrating the selective inhibition of cellular proliferation of thecancer cells.

Toxicity may also be measured based on cell viability, for example, theviability of cancer and normal cell cultures exposed to theimmunoconjugate may be compared. Cell viability may be assessed by knowntechniques, such as trypan blue exclusion assays.

In another example, a number of models may be used to test theeffectiveness of the immunoconjugates of the invention. Thompson, E. W.et al. (Breast Cancer Res. Treatment 31:357-370 (1994)) has described amodel for the determination of invasiveness of human breast cancer cellsin vitro by measuring tumor cell-mediated proteolysis of extracellularmatrix and tumor cell invasion of reconstituted basement membrane(collagen, laminin, fibronectin, Matrigel or gelatin). Other applicablecancer cell models include cultured ovarian adenocarcinoma cells (Young,T. N. et al. Gynecol. Oncol. 62:89-99 (1996); Moore, D. H. et al.Gynecol. Oncol. 65:78-82 (1997)), human follicular thyroid cancer cells(Demeure, M. J. et al., World J. Surg. 16:770-776 (1992)), humanmelanoma (A-2058) and fibrosarcoma (HT-1080) cell lines (Mackay, A. R.et al. Lab. Invest. 70:781 783 (1994)), and lung squamous (HS-24) andadenocarcinoma (SB-3) cell lines (Spiess, E. et al. J. Histochem.Cytochem. 42:917-929 (1994)). An in vivo test system involving theimplantation of tumors and measurement of tumor growth and metastasis inathymic nude mice has also been described (Thompson, E. W. et al.,Breast Cancer Res. Treatment 31:357-370 (1994); Shi, Y. E. et al.,Cancer Res. 53:1409-1415 (1993)).

The immunoconjugates of the invention may be formulated intopharmaceutical compositions for administration to subjects in abiologically compatible form suitable for administration in vivo. Thesubstances may be administered to living organisms including humans, andanimals. Administration of a therapeutically active amount of thepharmaceutical compositions of the present invention is defined as anamount effective, at dosages and for periods of time necessary toachieve the desired result. For example, a therapeutically active amountof a substance may vary according to factors such as the disease state,age, sex, and weight of the individual, and the ability of therecombinant protein of the invention to elicit a desired response in theindividual. Dosage regime may be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

Accordingly, the present invention provides a pharmaceutical compositionfor treating or preventing cancer comprising the immunoconjugates of theinvention, and a pharmaceutically acceptable carrier, diluent orexcipient. In a preferred embodiment, the effector molecule of theimmunoconjugate in the pharmaceutical composition is a cancertherapeutic agent, more preferably a toxin.

The pharmaceutical preparation comprising the immunoconjugate of theinvention may be administered systemically. The pharmaceuticalpreparation may be administered directly to the cancer site. Dependingon the route of administration, the immunoconjugate may be coated in amaterial to protect the compound from the action of enzymes, acids andother natural conditions that may inactivate the compound.

In accordance with one aspect of the present invention, theimmunoconjugate is delivered to the patient by direct administration.The invention contemplates the pharmaceutical composition beingadministered in at least an amount sufficient to achieve the endpoint,and if necessary, comprises a pharmaceutically acceptable carrier.

The invention also provides methods for reducing the risk ofpost-surgical complications comprising administering an effective amountof the immunoconjugate of the invention before, during, or after surgeryto treat cancer.

The compositions described herein can be prepared by per se knownmethods for the preparation of pharmaceutically acceptable compositionsthat can be administered to subjects, such that an effective quantity ofthe active substance is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., USA 1985). On thisbasis, the compositions include, albeit not exclusively, solutions ofthe substances in association with one or more pharmaceuticallyacceptable vehicles or diluents, and contained in buffered solutionswith a suitable pH and iso-osmotic with the physiological fluids.

Pharmaceutical compositions include, without limitation, lyophilizedpowders or aqueous or non-aqueous sterile injectable solutions orsuspensions, which may further contain antioxidants, buffers,bacteriostats and solutes that render the compositions substantiallycompatible with the tissues or the blood of an intended recipient. Othercomponents that may be present in such compositions include water,alcohols, polyols, glycerin and vegetable oils, for example.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules, tablets, or concentrated solutions orsuspensions. Immunoconjugate may be supplied, for example but not by wayof limitation, as a lyophilized powder which is reconstituted withsterile water or saline prior to administration to the patient.

Pharmaceutical compositions of the invention may comprise apharmaceutically acceptable carrier. Suitable pharmaceuticallyacceptable carriers include essentially chemically inert and nontoxiccompositions that do not interfere with the effectiveness of thebiological activity of the pharmaceutical composition. Examples ofsuitable pharmaceutical carriers include, but are not limited to, water,saline solutions, glycerol solutions, ethanol,N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA),diolesylphosphotidyl-ethanolamine (DOPE), and liposomes. Suchcompositions should contain a therapeutically effective amount of thecompound, together with a suitable amount of carrier so as to providethe form for direct administration to the patient.

The composition may be in the form of a pharmaceutically acceptable saltwhich includes, without limitation, those formed with free amino groupssuch as those derived from hydrochloric, phosphoric, acetic, oxalic,tartaric acids, etc., and those formed with free carboxyl groups such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

In various embodiments of the invention, the pharmaceutical compositionis directly administered systemically or directly to the area of thetumor(s).

The pharmaceutical compositions may be used in methods for treatinganimals, including mammals, preferably humans, with cancer. The dosageand type of immunoconjugate to be administered will depend on a varietyof factors which may be readily monitored in human subjects. Suchfactors include the etiology and severity (grade and stage) of thecancer.

Clinical outcomes of cancer treatments using the immunoconjugates of theinvention are readily discernable by one of skill in the relevant art,such as a physician. For example, standard medical tests to measureclinical markers of cancer may be strong indicators of the treatment'sefficacy. Such tests may include, without limitation, physicalexamination, performance scales, disease markers, 12-lead ECG, tumormeasurements, tissue biopsy, cytoscopy, cytology, longest diameter oftumor calculations, radiography, digital imaging of the tumor, vitalsigns, weight, recordation of adverse events, assessment of infectiousepisodes, assessment of concomitant medications, pain assessment, bloodor serum chemistry, urinalysis, CT scan, and pharmacokinetic analysis.Furthermore, synergistic effects of a combination therapy comprising theimmunoconjugate and another cancer therapeutic may be determined bycomparative studies with patients undergoing monotherapy.

Another embodiment of the invention is a kit for treating or preventingcancer comprising an effective amount of the immunoconjugate of theinvention, and directions for the use thereof to treat the cancer.

In the majority of approved anticancer therapies, the anticancer therapyis used in combination with other anticancer therapies. Accordingly, theinvention provides a method of preventing or treating cancer using theimmunoconjugate of the invention in combination with at least oneadditional anticancer therapy. The other cancer therapy may beadministered prior to, overlapping with, concurrently, and/or afteradministration of the immunoconjugate. When administered concurrently,the immunoconjugate and the other cancer therapeutic may be administeredin a single formulation or in separate formulations, and if separately,then optionally, by different modes of administration. The combinationof one or more immunoconjugates and one or more other cancer therapiesmay synergistically act to combat the tumor or cancer. The other cancertherapies include, without limitation, radiation and other anticancertherapeutic agents. These other cancer therapeutics may include, withoutlimitation, 2,2′,2″trichlorotriethylamine, 6-azauridine,6-diazo-5-oxo-L-norleucine, 6-mercaptopurine, aceglarone, aclacinomycinsactinomycin, altretamine, aminoglutethimide, aminoglutethimide,amsacrine, anastrozole, ancitabine, angiogenin antisenseoligonucleotide, anthramycin, azacitidine, azaserine, aziridine,batimastar, bcl-2 antisense oligonucleotide, benzodepa, bicalutamide,bisantrene, bleomycin, buserelin, busulfan, cactinomycin, calusterone,carboplatin, carboquone, caminomycin, carmofur, carmustine, carubicin,carzinophilin, chlorambucil, chlornaphazine, chlormadinone acetate,chlorozotocin, chromomycins, cisplatin, cladribine, cyclophosphamide,cytarabine, dacarbazine, dactinomycin, daunorubicin, defosfamide,demecolcine, denopterin, detorubicin, diaziquone, docetaxel,doxifluridine, doxorubicin, droloxifene, dromostanolone, edatrexate,eflomithine, elliptinium acetate, emitefur, enocitabune, epirubicin,epitiostanol, esorubicin, estramustine, etoglucid, etoposide, fadrozole,fenretinide, floxuridine, fludarabine, fluorouracil, flutamide, folinicacid, formestane, fosfestrol, fotemustine, gallium nitrate, gemcitabine,goserelin, hexestrol, hydroxyurea, idarubicin, ifosfamide, improsulfan,interferon-alpha, interferon-beta, interferon-gamma, interleukin-2,L-asparaginase, lentinan, letrozole, leuprolide, lomustine, lonidamine,mannomustine, marcellomycin, mechlorethamine, mechlorethamine oxidehydrochloride, medroxyprogesterone, megestrol acetate, melengestrol,melphalan, menogaril, mepitiostane, methotrexate, meturedepa,miboplatin, miltefo sine, mitobronitol, mitoguazone, mitolactol,mitomycins, mitotane, mitoxantrone, mopidamol, mycophenolic acid,nilutamide, nimustine, nitracine, nogalamycin, novembichin, olivomycins,oxaliplatin, paclitaxel, pentostatin, peplomycin, perfosfamide,phenamet, phenesterine, pipobroman, piposulfan, pirarubicin, piritrexim,plicamycin, podophyllinic acid 2-ethyl-hydrazide, polyestradiolphosphate, porfimer sodium, porfiromycin, prednimustine, procabazine,propagermanium, PSK, pteropterin, puromycin, quelamycin, ranimustine,razoxane, rodorubicin, roquinimex, sizofican, sobuzoxane,spirogermanium, streptonigrin, streptozocin, tamoxifen, taxotere,tegafur, temozolomide, teniposide, tenuzonic acid, testolacone,thiamiprine, thioguanine, thiotepa, Tomudex, topotecan, toremifene,triaziquone, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide, trilostane, trimetrexate, triptorelin,trofosfamide, trontecan, tubercidin, ubenimex, uracil mustard, uredepa,urethan, vinblastine, vincristine, zinostatin, and zorubicin, cytosinearabinoside, gemtuzumab, thioepa, cyclothosphamide, antimetabolites(e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil, fludarabine, gemcitabine, dacarbazine, temozoamide),hexamethylmelamine, LYSODREN, nucleoside analogues, plant alkaloids(e.g., Taxol, paclitaxel, camptothecin, topotecan, irinotecan(CAMPTOSAR, CPT-11), vincristine, vinca alkyloids such as vinblastine.)podophyllotoxin, epipodophyllotoxin, VP-16 (etoposide), cytochalasin B,gramicidin D, ethidium bromide, emetine, anthracyclines (e.g.,daunorubicin), doxorubicin liposomal, dihydroxyanthracindione,mithramycin, actinomycin D, aldesleukin, allutamine, biaomycin,capecitabine, carboplain, chlorabusin, cyclarabine, daclinomycin,floxuridhe, lauprolide acetate, levamisole, lomusline, mercaptopurino,mesna, mitolanc, pegaspergase, pentoslatin, picamycin, riuxlmab,campath-1, straplozocin, tretinoin, VEGF antisense oligonucleotide,vindesine, and vinorelbine. Compositions comprising one or more cancertherapeutics (e.g., FLAG, CHOP) are also contemplated by the presentinvention. FLAG comprises fludarabine, cytosine arabinoside (Ara-C) andG-CSF. CHOP comprises cyclophosphamide, vincristine, doxorubicin, andprednisone. For a full listing of cancer therapeutics known in the art,see, e.g., the latest editions of The Merck Index and the Physician'sDesk Reference.

Pharmaceutical compositions for combination therapy may also include,without limitation, antibiotics (e.g., dactinomycin, bleomycin,mithramycin, anthramycin), asparaginase, Bacillus and Guerin, diphtheriatoxin, procaine, tetracaine, lidocaine, propranolol, anti-mitoticagents, abrin, ricinA, Pseudomonas exotoxin, nerve growth factor,platelet derived growth factor, tissue plasminogen activator,antihistaminic agents, anti-nausea agents, etc.

Indeed, administration of an effective amount of an immunoconjugate to apatient in need of such treatment may result in reduced doses of anothercancer therapeutic having clinically significant efficacy. Such efficacyof the reduced dose of the other cancer therapeutic may not be observedabsent administration with an immunoconjugate. Accordingly, the presentinvention provides methods for treating a tumor or cancer comprisingadministering a reduced dose of one or more other cancer therapeutics.

Moreover, combination therapy comprising an immunoconjugate to a patientin need of such treatment may permit relatively short treatment timeswhen compared to the duration or number of cycles of standard treatmentregimens. Accordingly, the present invention provides methods, fortreating a tumor or cancer comprising administering one or more othercancer therapeutics for relatively short duration and/or in fewertreatment cycles.

Thus, in accordance with the present invention, combination therapiescomprising an immunoconjugate and another cancer therapeutic may reducetoxicity (i.e., side effects) of the overall cancer treatment. Forexample, reduced toxicity, when compared to a monotherapy or anothercombination therapy, may be observed when delivering a reduced dose ofimmunoconjugate and/or other cancer therapeutic, and/or when reducingthe duration of a cycle (i.e., the period of a single administration orthe period of a series of such administrations), and/or when reducingthe number of cycles.

Accordingly, the invention provides a pharmaceutical compositioncomprising an immunoconjugate and one or more additional anticancertherapeutic, optionally in a pharmaceutically acceptable carrier.

The present invention also provides a kit comprising an effective amountof an immunoconjugate, optionally, in combination with one or more othercancer therapeutic, together with instructions for the use thereof totreat cancer.

As stated above, combination therapy with an immunoconjugate maysensitize the cancer or tumor to administration of an additional cancertherapeutic. Accordingly, the present invention contemplates combinationtherapies for preventing, treating, and/or preventing recurrence ofcancer comprising administering an effective amount of animmunoconjugate prior to, subsequently, or concurrently with a reduceddose of a cancer therapeutic. For example, initial treatment with animmunoconjugate may increase the sensitivity of a cancer or tumor tosubsequent challenge with a dose of cancer therapeutic. This dose isnear, or below, the low range of standard dosages when the cancertherapeutic is administered alone, or in the absence of animmunoconjugate. When concurrently administered, the immunoconjugate maybe administered separately from the cancer therapeutic, and optionally,via a different mode of administration.

Accordingly, in one embodiment, the additional cancer therapeuticcomprises cisplatin, e.g., PLATINOL or PLATINOL-AQ (Bristol Myers), at adose ranging from approximately 5 to 10, 11 to 20, 21 to 40, or 41 to 75mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisescarboplatin, e.g., PARAPLATIN (Bristol Myers), at a dose ranging fromapproximately 2 to 3, 4 to 8, 9 to 16, 17 to 35, or 36 to 75mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisescyclophosphamide, e.g., CYTOXAN (Bristol Myers Squibb), at a doseranging from approximately 0.25 to 0.5, 0.6 to 0.9, 1 to 2, 3 to 5, 6 to10, 11 to 20, or 21 to 40 mg/kg/cycle.

In another embodiment, the additional cancer therapeutic comprisescytarabine, e.g., CYTOSAR-U (Pharmacia & Upjohn), at a dose ranging fromapproximately 0.5 to 1, 2 to 4, 5 to 10, 11 to 25, 26 to 50, or 51 to100 mg/m²/cycle. In another embodiment, the additional cancertherapeutic comprises cytarabine liposome, e.g., DEPOCYT (Chiron Corp.),at a dose ranging from approximately 5 to 50 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesdacarbazine, e.g., DTIC or DTICDOME (Bayer Corp.), at a dose rangingfrom approximately 15 to 250 mg/m²/cycle or ranging from approximately0.2 to 2 mg/kg/cycle.

In another embodiment, the additional cancer therapeutic comprisestopotecan, e.g., HYCAMTIN (SmithKline Beecham), at a dose ranging fromapproximately 0.1 to 0.2, 0.3 to 0.4, 0.5 to 0.8, or 0.9 to 1.5mg/m²/Cycle. In another embodiment, the additional cancer therapeuticcomprises irinotecan, e.g., CAMPTOSAR (Pharmacia & Upjohn), at a doseranging from approximately 5 to 9, 10 to 25, or 26 to 50 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesfludarabine, e.g., FLUDARA (Berlex Laboratories), at a dose ranging fromapproximately 2.5 to 5, 6 to 10, 11 to 15, or 16 to 25 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisescytosine arabinoside (Ara-C) at a dose ranging from approximately 200 to2000 mg/m²/cycle, 300 to 1000 mg/m²/cycle, 400 to 800 mg/m²/cycle, or500 to 700 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesdocetaxel, e.g., TAXOTERE (Rhone Poulenc Rorer) at a dose ranging fromapproximately 6 to 10, 11 to 30, or 31 to 60 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisespaclitaxel, e.g., TAXOL (Bristol Myers Squibb), at a dose ranging fromapproximately 10 to 20, 21 to 40, 41 to 70, or 71 to 135 mg/kg/cycle.

In another embodiment, the additional cancer therapeutic comprises5-fluorouracil at a dose ranging from approximately 0.5 to 5mg/kg/cycle, 1 to 4 mg/kg/cycle, or 2-3 mg/kg/cycle.

In another embodiment, the additional cancer therapeutic comprisesdoxorubicin, e.g., ADRIAMYCIN (Pharmacia & Upjohn), DOXIL (Alza), RUBEX(Bristol Myers Squibb), at a dose ranging from approximately 2 to 4, 5to 8, 9 to 15, 16 to 30, or 31 to 60 mg/kg/cycle.

In another embodiment, the additional cancer therapeutic comprisesetoposide, e.g., VEPESID (Pharmacia & Upjohn), at a dose ranging fromapproximately 3.5 to 7, 8 to 15, 16 to 25, or 26 to 50 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesvinblastine, e.g., VELBAN (Eli Lilly), at a dose ranging fromapproximately 0.3 to 0.5, 0.6 to 0.9, 1 to 2, or 3 to 3.6 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesvincristine, e.g., ONCOVIN (Eli Lilly), at a dose ranging fromapproximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesmethotrexate at a dose ranging from approximately 0.2 to 0.9, 1 to 5, 6to 10, or 11 to 20 mg/m²/cycle.

In another embodiment, an immunoconjugate is administered in combinationwith at least one other immunotherapeutic which includes, withoutlimitation, rituxan, rituximab, campath-1, gemtuzumab, and trastuzutmab.

In another embodiment, an immunoconjugate is administered in combinationwith one or more anti-angiogenic agents which include, withoutlimitation, angiostatin, thalidomide, kringle 5, endostatin, Serpin(Serine Protease Inhibitor), anti-thrombin, 29 kDa N-terminal and a 40kDa C-terminal proteolytic fragments of fibronectin, 16 kDa proteolyticfragment of prolactin, 7.8 kDa proteolytic fragment of plateletfactor-4, a 13 amino acid peptide corresponding to a fragment ofplatelet factor-4 (Maione et al., 1990, Cancer Res. 51:2077-2083), a14-amino acid peptide corresponding to a fragment of collagen I (Tolmaet al., 1993, J. Cell Biol. 122:497-511), a 19 amino acid peptidecorresponding to a fragment of Thrombospondin I (Tolsma et al., 1993, J.Cell Biol. 122:497-511), a 20-amino acid peptide corresponding to afragment of SPARC (Sage et al., 1995, J. Cell. Biochem. 57:1329-1334),and a variant thereof, including a pharmaceutically acceptable saltthereof.

In another embodiment, an immunoconjugate is administered in combinationwith a regimen of radiation therapy. The therapy may also comprisesurgery and/or chemotherapy. For example, the immunoconjugate may beadministered in combination with radiation therapy and cisplatin(Platinol), fluorouracil (5-FU, Adrucil), carboplatin (Paraplatin),and/or paclitaxel (Taxol). Treatment with the immunoconjugate may allowuse of lower doses of radiation and/or less frequent radiationtreatments, which may for example, reduce the incidence of severe sorethroat that impedes swallowing function potentially resulting inundesired weight loss or dehydration.

In another embodiment, an immunoconjugate is administered in combinationwith one or more cytokines which include, without limitation, alymphokine, tumor necrosis factors, tumor necrosis factor-like cytokine,lymphotoxin, interferon, macrophage inflammatory protein, granulocytemonocyte colony stimulating factor, interleukin (including, withoutlimitation, interleukin-1, interleukin-2, interleukin-6, interleukin-12,interleukin-15, interleukin-18), and a variant thereof, including apharmaceutically acceptable salt thereof.

In yet another embodiment, an immunoconjugate is administered incombination with a cancer vaccine or biological agents including,without limitation, autologous cells or tissues, non-autologous cells ortissues, carcinoembryonic antigen, alpha-fetoprotein, human chorionicgonadotropin, BCG live vaccine, Mycobacterial cell wall-DNA complexes,melanocyte lineage proteins, and mutated, tumor-specific antigens.

In yet another embodiment, an immunoconjugate is administered inassociation with hormonal therapy. Hormonal therapeutics include,without limitation, a hormonal agonist, hormonal antagonist (e.g.,flutamide, tamoxifen, leuprolide acetate (LUPRON)), and steroid (e.g.,dexamethasone, retinoid, betamethasone, cortisol, cortisone, prednisone,dehydrotestosterone, glucocorticoid, mineralocorticoid, estrogen,testosterone, progestin).

In yet another embodiment, an immunoconjugate is administered inassociation with a gene therapy program to treat or prevent cancer.

Combination therapy may thus increase the sensitivity of the cancer ortumor to the administered immunoconjugate and/or additional cancertherapeutic. In this manner, shorter treatment cycles may be possiblethereby reducing toxic events. The cycle duration may vary according tothe specific cancer therapeutic in use. The invention also contemplatescontinuous or discontinuous administration, or daily doses divided intoseveral partial administrations. An appropriate cycle duration for aspecific cancer therapeutic will be appreciated by the skilled artisan,and the invention contemplates the continued assessment of optimaltreatment schedules for each cancer therapeutic. Specific guidelines forthe skilled artisan are known in the art. See, e.g., Therasse et al.,2000, “New guidelines to evaluate the response to treatment in solidtumors. European Organization for Research and Treatment of Cancer,National Cancer Institute of the United States, National CancerInstitute of Canada,” J Natl Cancer Inst. February 2; 92(3):205-16.

It is contemplated that the immunoconjugate may be administered by anysuitable method such as injection, oral administration, inhalation,transdermal or intratumorally, whereas any other cancer therapeutic maybe delivered to the patient by the same or by another mode ofadministration. Additionally, where multiple cancer therapeutics areintended to be delivered to a patient, the immunoconjugate and one ormore of the other cancer therapeutics may be delivered by one method,whereas other cancer therapeutics may be delivered by another mode ofadministration.

(F) Diagnostic Methods and Agents

The binding proteins of the invention bind selectively to cancer cellsor molecules internalized by cancer cells, and not significantly tonormal cells. Therefore the binding proteins can be used in thediagnosis of cancer. As stated above, the inventors have shown that thebinding proteins of the invention binds to the extracellular domain ofCD44E. The inventors have also shown that the binding proteins of theinvention bind to AFP or a variant thereof. AFP is associated withabnormal growth, cell transformation and cancer. Thus, the specificityof the binding proteins for tumor antigens makes it useful in thediagnosis of cancer.

In a preferred embodiment, the binding proteins are antibodies orantibody fragments of the invention. In addition, cancer cells may beevaluated to determine their susceptibility to the treatment methods ofthe invention by, for example, obtaining a sample of the cancer cellsand determining the ability of the sample to bind to the bindingproteins of the invention, preferably antibodies or antibody fragments.

Accordingly, the present invention includes diagnostic methods, agents,and kits that can be used by themselves or prior to, during orsubsequent to the therapeutic method of the invention in order todetermine whether or not cancer cells are present that express theantigen and can bind to the binding proteins of the invention,preferably antibodies and antibody fragments.

In one embodiment, the invention provides a method of diagnosing cancerin a mammal comprising the steps of

-   -   (1) contacting a test sample taken from said mammal with the        binding proteins of the invention that binds to an antigen on or        in the cancer cell under conditions that permit the formation of        a binding protein-antigen complex;    -   (2) measuring the amount of binding protein-antigen complex in        the test sample; and    -   (3) comparing the amount of binding protein-antigen complex in        the test sample to a control.

In one embodiment, the antigen is CD44E; a protein having a molecularweight between 47-53 kDa and an isoelectric point between 5.2-5.5,preferably 5.4; or a protein comprising the 5-v8 interface of CD44E, v8exon of CD44 or the amino acid sequence ATNMDSSHSIT. In anotherembodiment, the antigen is alpha-fetoprotein or a variant thereof; aprotein having a molecular weight between 48-54 kDa and an isoelectricpoint between 5.1-5.4, preferably 5.2; or a protein comprising aminoacid SEQ ID NOS: 14, 15 or 16. In another example, the antigen is aprotein comprising amino acid SEQ ID NOS: 38, 39, 40, 41, 42, 43, 44 or45 and has a molecular weight between 47-53 kDa and an isoelectric pointbetween 5.2-5.5; or a protein comprising amino acid SEQ ID NOS: 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74 or 75 and has a molecular weightbetween 48-54 kDa and an isoelectric point between 5.1-5.4.

Another embodiment of the invention is a method of diagnosing cancer ina mammal comprising the steps:

-   -   (4) contacting a test sample from said mammal with an antibody        that binds to alpha-fetoprotein or a variant thereof under        conditions that permit the formation of an        antibody-alpha-fetoprotein complex and an antibody that binds to        CD44E under conditions that permit the formation of an        antibody-CD44E complex;    -   (5) measuring the amount of antibody-alpha-fetoprotein complex        and antibody-CD44E complex in the test sample; and    -   (6) comparing the amount of antibody-alpha-fetoprotein complex        and antibody-CD44E complex in the test sample to a control.

The invention further includes a kit for diagnosing cancer comprisingany one of the binding proteins of the invention and instructions forthe use thereof to diagnose the cancer. The invention also includes akit for diagnosing cancer comprising an antibody that binds toalpha-fetoprotein and an antibody that binds to CD44E and instructionsfor the use thereof to diagnose cancer.

For use in the diagnostic applications, the binding proteins of theinvention, preferably antibodies or antibody fragments, may be labeledwith a detectable marker such as a radio-opaque or radioisotope, such as³H, ¹⁴C, ³²P, ³⁵S, ¹²³I, ¹²⁵I, ¹³¹I; a fluorescent (fluorophore) orchemiluminescent (chromophore) compound, such as fluoresceinisothiocyanate, rhodamine or luciferin; an enzyme, such as alkalinephosphatase, beta-galactosidase or horseradish peroxidase; an imagingagent; or a metal ion. As described above, methods of attaching a labelto a binding protein, such as an antibody or antibody fragment, areknown in the art.

Another aspect of the invention is a method of diagnosing cancer in amammal comprising the steps of

-   -   (1) measuring the amount of antibodies of the invention in a        test sample taken from said mammal; and    -   (2) comparing the amount of antibodies of the invention in the        test sample to a control.

In one embodiment, the amount of antibodies of the invention is measuredby measuring the amount of antibodies of the invention in the testsample, for example by ELISA. In another embodiment, the amount ofantibodies of the invention is measured by measuring the expressionlevels of nucleic acids encoding the antibodies of the invention in thetest sample, for example by RT-PCR.

(G) Antigens

As mentioned above, the inventors have identified the antigen of thebinding proteins of the invention. Accordingly, the invention includesan isolated protein that can specifically bind with one of the bindingproteins of the invention, and nucleic acid sequences and uses thereof.

In one example, the isolated protein has a molecular weight between47-53 kDa and an isoelectric point between 5.2-5.5, preferably 5.4; aprotein having a molecular weight between 48-54 kDa and an isoelectricpoint between 5.1-5.4, preferably 5.2; or a protein comprising the aminoacid sequence 107 to 487 of AFP (SEQ ID NO:14), 107 to 590 of AFP (SEQID NO: 15) or 107 to 609 of AFP (SEQ ID NO: 16). In another example, theisolated protein comprises amino acid SEQ ID NOS: 38, 39, 40, 41, 42,43, 44 or 45 and has a molecular weight between 47-53 kDa and anisoelectric point between 5.2-5.5; or comprises SEQ ID NOS: 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74 or 75 and has a molecular weight between48-54 kDa and an isoelectric point between 5.1-5.4, preferably 5.2.

(H) Other Uses of the Binding Proteins of the Invention

Antibodies to CD44 have been shown to block the PMA-induced binding ofCD44H (the standard form, also called CD44s) and CD44E to hyaluronicacid (HA) (Liao et al. J Immunol 151(11):6490-99, 1993). Clustering ofCD44 variants, particularly those that contain variant exon 9 appears tobe important for binding to HA and can be induced by PMA. Down streamintracellular signaling is related to this clustering and interferingwith it can affect cell function (Suzuki et al., JBC 277(10):8022-32,2002). It is possible that the blocking effect of antibodies on HAbinding is mediated by interference with clustering. Regardless of themechanism, the binding proteins of the invention could be used tomodulate the binding of CD44 to the extracellular molecules and thedownstream cell signaling resulting from clustering, or the binding toHA or/or other extracellular molecules.

Accordingly, the invention includes the use of the binding proteins ofthe invention to modulate the activity of CD44E. For example, thebinding proteins of the invention can be used to interfere with thebinding of CD44E to HA. The binding proteins of the invention may alsobe used to enhance CD44E activity.

The following non-limiting examples are illustrative of the presentinvention:

EXAMPLES Example 1 Generation of VB1-008 Monoclonal Antibody

The VB1-008 monoclonal antibody was generated from the peripheral bloodlymphocytes of a breast cancer patient. TM-SH-P2 was used as the fusionpartner to generate the monoclonal antibody. VB1-008 is an IgG1, lambdamonoclonal antibody.

Messenger RNA (mRNA) was isolated from hybridoma cells and first strandcomplement DNA (cDNA) was synthesized. The cDNA was then used to isolateantibody H and L chain genes by PCR. PCR primers were designed (seenote) according to the consensus framework regions of the H (Gamma) andL (Lambda) chain isotypes. The PCR products were individually clonedinto the TOPO-pCR 2.1 vector and transformed into E. coli cells.Individual clones containing the inserts in TOPO-pCR 2.1 were isolatedand grown. Plasmid DNA was purified and sequenced.

Gamma Primers: (SEQ ID NO: 18) 1) 5′TCT AAA GAA GCC CCT GGG AGC ACA GCT CAT CAC CAT G 3′ (SEQ ID NO: 19) 2)5′ GCC CGG GGA GCG GGG GCT TGC CGG CCG TCG CAC TCA 3′ (SEQ ID NO: 20) 3)5: ACC ATG AGT GAG AAA AAC TGG ATT TGT GTG GCA 3^(J) (SEQ ID NO: 21) 4)5′ GGA GCC GGT GAC CAG GGT TCC CTG GCC CCA 3′ (SEQ ID NO: 22) 5) 5′CTC ACC ATG GAG TTT GGG CTG AGC TGG GTT 3′ (SEQ ID NO: 23) 6) 5′GGA GGC TGA GGA GAC GGT GAC CAG GGT TCC CTG GCC 3′ Lambda Primers:(SEQ ID NO: 24) 7) 5′ GGC TCG AGA TGR CCT GSW CYC CTC TCY TYC TSW YC 3′(SEQ ID NO: 25) 8) 5′ CCC GTC GAC GAA GCT CCT TCA GAG GAG GG 3′ *

Note: In order to isolate as many varieties as possible using a singleprimer, mixed bases are used for certain consensus primers: R=A+G,D=A+T+G, Y=C+T, H=A+C+T, V=A+C+G, K=T+G, S=C+G, W=A+T.

Each PCR reaction comprised the following components in a 50 uL reactionvolume.

-   -   10×PCR buffer 5 μL    -   2 mM dNTPs 5 μL    -   50 mM MgCl2 2 μL    -   5′ Primer 20 pmoL    -   3′ Primer 20 pmoL    -   Taq DNA Polymerase 2.5 U    -   DNA template 50 ng

The PCR cycling conditions were: 94° C. for 1 min., 62° C. for 1 min.,72° C. for 1.5 min. for 30 cycles and a final extension for 10 min. at72° C. Amplified PCR products were electrophoretically separated on a 1%agarose gel, excised, purified using a Qiaquick gel extraction kit,cloned into the TOPO pCR 2.1 cloning vector and then DNA sequenced usingthe 373 DNA sequencer stretch (Griffin G. H. and Griffin M A: PCRtechnology, Current innovations. CRC Press, Boca Raton Fla. 3431 USA;Cloning vector pCR 2.1, Catalogue #205184. Invitrogen, Carlsbad, Calif.;Qiagen, Qiaquick gel extraction kit, Catalogue #28706. Qiagen Inc.,Mississauga, ON; and 373 DNA Stretch. PE Applied Biosystems, MississaugaON.).

The CDR sequences for VB1-008 are shown in Table 1.

The light chain variable region and the heavy chain variable region areshown in FIGS. 1 and 2, respectively.

Example 2 Antibody Profiling by Measuring Tumor Cell Reactivity

VB1-008 was tested by flow cytometry for tumor cell reactivity againsttwo panels of cell lines. The first panel comprises fifteen differenttypes of epithelial cancers while a second panel consists of five typesof normal cells. The VB1-008 results are summarized in Table 2. VB1-008had an MF>2.0 for all cancer types tested. MF values indicate the meancalculated from the sum of the mean fold increase in median fluorescenceover the control antibody from all cell lines in each indication. Thestrongest indications were, but not limited to, breast, lung, melanomaand prostate. In comparison, VB1-008 was more reactive with most of thetumor cell lines than with the normal cell lines. The two exceptionswere the kidney and lung cell lines; however, they were still lower thanthe corresponding tumor cell type. See Table 2. The fold-increase inVB1-008 reactivity of tumor: normal varied from ˜2 to 7.

Example 3 Normal Tissue Microarray

VB1-008 was tested against the flow positive tumor cell line SKBR-3 toassess the appropriate tissue format to demonstrate membrane stainingand to define the optimal conditions for staining. This antibodydemonstrated cytoplasmic and cell membrane staining in all theexperimental groups, including fixed embedded cells. In fixed cellpellets incubated overnight with VB1-008, 80% of the cells showedcytoplasmic staining, and 10% of them showed cell membrane staining.Representative pictures of cell membrane staining of formalin-fixed cellpellet cores are shown in FIG. 3.

Once the optimal staining conditions were identified, the antibody wastested in comparison with an isotype control (4B5) on a low density (LD)array of critical normal for normal tissue reactivity. The results forVB1-008 are summarized in Table 3. No significant membrane staining ofany of the normal critical tissues was observed. High density (HD) arraystaining of non-critical normal tissue showed that cell surface stainingwas limited to epithelial cells associated with reproduction-relatedtissues (testis and fallopian tubes, FIG. 4, Table 4). Otherwise, nosignificant staining was observed of any of the tissues was observed.

Example 4 Tumor Tissue Microarray

VB1-008 was tested in a HD formalin-fixed tumor TMA for tumor tissuereactivity. See Table 5. VB1-008 exhibited moderate cell surfacereactivity against a wide variety of indications including, bladder,breast, colon, kidney, liver, ovary, prostate, rectum, stomach anduterus. VB1-008 cell surface binding was lesser represented and at alower reactivity with cancers of the cervix, lung, pancreas, and skin.Representative pictures illustrating the cell surface reactivity VB1-008but not the isotype-matched control antibody to some of the cancers areshown in FIGS. 5-7.

Example 5 Assessment of VB1-008 Binding and Internalization by FlowCytometry and Confocal Microscopy

VB1-008 and two control antibodies (5E9 and MA-103) that demonstratestrong reactivity against the tumor cell line A-375 were used to assessVB1-008 for internalization. A representative experiment is shown inTable 6. VB1-008 binding results at different temperatures were notdifferent from the internalizing antibody 5E9. After 60 min at 37° C.,the membrane-bound VB1-008 disappeared from the cell surface, with a57.5% reduction in median fluorescence. Increasing the incubation timeat 37° C. was associated with a further decline in median fluorescence.By 120 min, the median fluorescence had decreased by 62.2%. Flowhistograms demonstrating cell-surface binding are illustrated in FIG. 8.To confirm if the cell-surface bound VB1-008 internalized into A-375cells or instead was shed from the plasma membrane, antibody-treatedcells were further evaluated by direct visualization of fluorescencedistribution and intracellular staining with the aid of laser scanningconfocal microscopy. Like MA-103 and 5E9, incubation of A-375 cells withVB1-008 at 4° C. for 60 min demonstrated a circumferential surfacedistribution of fluorescence label (FIG. 9A). Warming the VB1-008antibody bound cells to 37° C. revealed a punctuated pattern ofintracellular staining by the internalized antibody within 60 minutes,as shown in FIG. 9B.

Example 6 Binding Affinity

Flow cytometry was used to assess functional affinity [Benedict, C. A.,NacKrell, A. J. and Anderson, W. F. (1997) J. Immunol. Methods,201:223-231]. A range of antibody concentrations were tested against afixed number of tumor cells (A-375) for 2-hours to construct asaturation curve. Values and graphical analysis were generated usingSigma Plot (Jandel Scientific, San Rafael, Calif.). The inverse of thedetermined median fluorescence was plotted as a function of the inverseof antibody concentration to determine KD by the Lineweaver-Burk method.A straight line was generated and the KD was calculated from the slopeof the curve. The dissociation constant KD values were determined by thefollowing equation: 1/F=1/Fmax+(KD/Fmax)(1/IgG or IgM or scFv), whereF=background subtracted median fluorescence and Fmax was calculated fromthe plot. The dissociation constant for VB1-008 was shown to be5.88×10⁻⁸M.

Example 7 VB1-008 Antigen Identification Cells

Breast cancer cell lines, MDA-MB 435S, MDA-MB-231; MCF-7; melanoma cellline, A-375; pancreatic tumor cell line, PANC-1 and T-cell lines, Daudiand Ramos were used in the study (Table 7). These cell lines wereselected based on the results of tumor cell line profiling by flowcytometry.

Growth and Maintenance of Tumor Cell Lines

The cell lines in the study were purchased from ATCC and cultured inaccordance with the guidelines and recommendations of ATCC. Cells wereharvested at 90% confluence with viability >90%.

Preliminary Characterization of the Antigen Binding to VB1-008

Preliminary characterization data was obtained from experiments designedto assess the feasibility of the gel-based approach by dot blot assays;and from experiments performed to determine the nature of the epitopeassociated with the antigens.

The data from these experiments classified the VB1-008 antigen as a“blottable” antigen with a peptide epitope, i.e., the epitope involvedin binding to VB1-008 on the antigen was neither glycosylated nor lipidassociated. It should be noted that the antigen could be glycosylated atsites other than the binding site.

VB1-008 Ag Enrichment and Purification

Immunoprecipitation

A minimum of 500 μg membrane protein was used for immuno-affinitypurification. A pre-clearing step using protein-G sepharose alone wasthe first step in the purification of the antigen prior to the additionof the antibody. In certain cases, pre-clearing was performed twice toadd more stringency to the assay. A total of 15-20 μg of antibody wasused as the precipitating agent in the mixture. The antigen-antibodymixtures were mutated overnight at 4° C. using buffer conditions thatmimicked physiologic conditions. Care was taken to ensure that proteaseinhibitors were used in every step of the antigen isolation process.

Immune complexes were centrifuged, washed with RIP-A lysis buffer andeluted with 0.2 M glycine pH 2.5. Supernatants representing the unboundfractions were stored to test the proteins that were not isolated byaffinity purification. Immunoprecipitations were carried out on two verypositive cell lines, i.e., A-375 and MDA-MB-435S, one moderatelypositive cell line, MDA-MB-231; one weakly positive cell line, i.e.,MCF-7; and three negative cell lines, i.e., Panc-1; Daudi and Ramos,with VB1-008 and equal amounts of 4B5 (isotype-matched control)processed in parallel at all times.

Gel-Based Analysis and Western Blotting

1D-PAGE

The purified proteins were subjected to reducing and non-reducingconditions of sample preparation and were subsequently analyzed bySDS-PAGE/Western Blotting. When reducing conditions were used, theisolated antigens were treated with sample buffer containing 1%|3-mercaptoethanol at 65° C. for 15 minutes and when non-reducingconditions were used, the antigens were mixed with sample buffer devoidof any reducing agent. The resulting blots were probed with the requiredantibodies and corresponding secondary antibodies conjugated to HRP, tovisualize the immuno-purified proteins by chemiluminescence.

2D-PAGE

The immunoprecipitated proteins were separated by two-dimensional gelelectrophoresis to resolve any protein stacking effect that may haveoccurred in the 1D-PAGE analysis. The 2D-gel electrophoresis resolvedproteins according to their isoelectric points (Pi) in the firstdimension and on the basis of their molecular weights in the seconddimension. The proteins thus resolved were transferred to nitrocellulosemembranes, overnight, and processed as in the case of 1D-PAGE. Westernblots were probed with VB1-008, anti-CD44 and anti-AFP as required andreacting proteins visualized by chemiluminescence.

Peptide Extraction and Antigen ID

The proteins were excised from 1D-gel and 2D-gels and analyzed. Raw datawas obtained predicting the probable proteins based on the number ofpeptides received. The LC-MS/MS runs were carried out on ‘QSTAR- andLCQ-dodeca LC-MS/MS from Thermo Finnigan. De-novo sequencing of theidentified proteins was also performed at the same facility.

Example 7(a) 1D-PAGE/Western Analysis

Only one specific band was detected after separation on a 1D-PAGE at˜110 kDa under non-reducing conditions (FIG. 10A) in antigen-positivecell lines (A-375, MDA-MB-435S). The same band was weakly detected inthe weakly positive cell lines (MCF-7) and absent in theantigen-negative cell line (Daudi). When samples were separated onSDS-PAGE under reducing conditions of sample preparation, a predominantband at ˜50 kDa and a faint 110 kDa band were observed expressedstrongly in antigen-positive cell lines, MDA-MB-4355, A-375, MDA-MB-231,weakly expressed in MCF-7, and absent in antigen-negative cell lines,such as Daudi and Panc-1 (FIG. 10B; FIG. 10C); Ramos was an exception tothe above observations (FIGS. 11B and 10C). None of the cell linesshowed positive immunoprecipitation with 4B5. The Western data issummarized in Table 8.

To determine the specificity of binding of the antigens detected by IPand Western blotting, four cell lines were pre-cleared twice and theresulting solutions immunoprecipitated with VB1-008. As can be seen inFIG. 10B, no band was detected in MCF-7, but the rest of the cell lines,showed the same 2 specific bands at ˜50 kDa and ˜110 kDa (faint). Apartfrom these, as seen in FIG. 10A as well, immunoprecipitation with 4B5did not yield any detectable reactive proteins with VB1-008, indicatingspecificity in the purification technique employed. The binding profilesof VB1-008 to these seven cell lines, measured by flow cytometry, werecomparable to the results observed in the immunopurification experiments(Table 8).

Example 7(b) 2D-PAGE Analysis

In order to determine isoelectric points (Pi) and assess the possibilityof protein stacking in the 1D-PAGE analysis, the purified antigens forVB1-008 were separated on two-dimensional polyacrylamide gelelectrophoresis (20-PAGE), where the separation in the first dimensionwas on the basis of Pi and the second dimension on the basis ofmolecular weight. The gels were then transferred to nitrocellulosemembranes and subjected to standard Western blotting processing. Sincethe amounts required for the detection of proteins on a 2D gel is ˜4times higher than the requirement for a 1D gel, purified antigens from 4separate immunoprecipitation reactions were pooled together for one2D-PAGE analysis. Two separate gels were processed simultaneously forWestern blot analysis to ensure that the proteins detected on theCoomassie stained gels were the same as those observed in the Westernblots. The 2D Western blots were probed with VB1-008 and detected by ECL(chemiluniscence). As can be seen in FIG. 11A, two spots were detectedat ˜49 kDa/Pi=5.2-5.6.

FIG. 11B represents the coomassie stained profile of theimmunoprecipitates from MDA-MB-435S separated by two-dimensional gelelectrophoresis. The two spots that were observed, labeled as spots “C”and “D” were excised for MS analysis. The details of the proteinsidentified are given in the Tables 9A and 9B, respectively.

Peptide Extraction and Protein Analysis

A-375 and MDA-MB-435S membranes were used to immunopurify antigen(s)that bind specifically to VB1-008. Under reducing conditions of gelseparation, ˜50 kDa band was observed in both the cell lines and undernon-reducing conditions, ˜110 kDa band was observed, referred to as “E”from MDA-MB-435S cells. These protein bands were excised from thecoomassie stained gels for MS analysis.

Proteins from 1D-gel band and 2D-spots were digested with trypsin torelease them from the gel and analyzed on a reverse-phase LC-MS/MSsystem. The identities of the proteins were revealed by databaseanalysis using bioinformatic tools. Raw data included peptides obtained,and a list of suggested proteins including contaminants such as keratin.To obtain the analysis MS/MS spectra were submitted directly to Mascotsearch engines available at www.Matrixscience.com.

Analysis of Peptide Masses and their Identities

The connection between the isoelectric point (Pi) and the molecularweight of the putative protein candidate is a critical parameter forprotein ID. Care was taken during analysis to ensure that the identifiedpeptide masses and their Pi were within ±3 kDa range and ±0.2 Pirespectively. This is because of the inherent possibility of peptides toexist in different modified states, resulting in their deviation fromthe theoretically calculated masses and Pi. Any acceptable deviationshould not be more than the values specified earlier. In cases, wherethe number of peptides was very low, an additional MS step was requiredto obtain more information by a process known as “de-novo sequencing”.De-novo sequencing is a process where a second MS step fragments each ofthe peptides obtained in the first MS run into peptide fragment ions (yand b ions), each representing an ionized form of an amino acid. Thesequence of each peptide can then be deduced from the resulting massspectrum.

Peptides have a general tendency to undergo modifications such asoxidation of methionines; esterification of acidic “R” groups, acetamideformations of amine groups and hydroxylations of proline, hydroxyprolineand glycine residues during MS/MS fragmentations. When thesemodifications occur, the peptide masses, although identical areperceived as different peptides, resulting in a false increase inscoring pattern of the protein ID, which is otherwise a cumulative unitof all the individual peptides identified. If the peptides are notanalyzed properly, spurious scores may arise leading to incorrectprotein identification. Therefore, it was critical to assess and select“unique” peptides that were not repetitive or represented elsewhere andaward scores correctly on the basis of these unique peptides. Inaddition, several other parameters such as the SE window, the number ofmissed cleavages, metastable fragmentation, single amino acidmodifications, etc., were taken into account before the final analysiswas performed in-house. As a consequence of these stringent steps, alarge number of peptides were drastically reduced to a fewer number. Thedatabase searches using these edited lists pulled down mapped proteins.Since the procedure employed here is immunopurification, the presence ofremnant antibody also was considered as a contaminant along withwell-known contaminants such as actin, vimentin, keratin, cytokeratinand tubulin. The resulting 3-4 final proteins were legitimate IDs,selected or eliminated based on the Pi and molecular weights of theproteins deduced by 2D-PAGE.

Analysis of 2D Spot “C”

Spot “C” excised from the 2D-gel identified only alpha-fetoprotein(AFP), while the other two proteins listed were protease inhibitorsadded for the integrity of the protein during the study. The Pi alsomatches the possibility of the molecule being AFP. The MS analysisrevealed 65 peptides, but only 30 unique peptides were retrieved whichconstituted 54% sequence coverage for human AFP with each peptideshowing 100% homology to the original protein. However, the AFP moleculelacked the first 160 aa from the N-terminus. Sequence analysis of thehuman AFP molecule showed clear presence of lysine and arginine residuesin these first 106 aa, which could be cleaved as peptides, should theybe present in the molecule. De-novo sequencing information of the 2Dspot “C”, showed a lack of 160 aa from the N-terminus, which has been arecurrent phenomenon when the identity of AFP was established (FIG.12A). The combined results of De-novo sequencing from the 1D gel and the2D gel is shown in FIG. 12B. The results show a lack of 106 aa from theN-terminus. Table 11A lists the peptides identified.

Analysis of 2D-Spot “D”

Spot “D” from the 2D-gel revealed the identities of 3 proteins inaddition to co-purifying contaminants, actin and actin-binding proteinactinin. However, except for CD44, the Pi of the other two proteins weredistinctly different from the one observed for the 2D spot, thereforethey were excluded as protein IDs. The molecular weight of the CD44isoform 3 was determined to be 53.585±3 kDa making it a complete matchfor the molecular weight and Pi observed on 2D-PAGE analysis for thespot “D”.

Analysis of the 110 kDa Antigen Band

As mentioned earlier, under reducing conditions the ˜110 kDa band wasvisualized by both Coomassie and Western blot analysis. From the 20-PAGEanalysis, it was clear that there were two components each around ˜50kDa, individually identified as CD44 and AFP, contributing together toform a 110 kDa band when the conformation was preserved undernon-reducing conditions of gel separation. Thus for confirmation, the110 kDa band was excised and analyzed to identify the proteincomponents. The ˜110 kDa band seen in FIG. 13A, was excised (E) for MSanalysis. The details of the proteins identified from the 100 kDa bandare given in Table 10.

MS Analysis of Protein Band “E”

The results of the MS analysis for protein band “E” are given in Table10. Apart from the co-purifying contaminants, i.e., actin, actinin andvimentin, three protein identities were obtained. Among them were CD44,AFP and heat shock protein 90. Heat shock protein 90 was not a match forthe molecular weight identified, and was therefore excluded as apotential candidate. Since CD44 is membrane-associated, it is likely thecognate antigen. It has also been demonstrated that AFP co-purifies withCD44 (FIG. 15A), however, AFP was not detected on the membrane surface.

Using top-down proteomics approach, it was clear that the molecularweight of the isolated antigen (50 kDa) corresponded to the predictedmolecular weight of CD44E. Flow experiments and the binding rank orderto the given cell lines also validate this finding. Data in Tables 11Band 12 describe the details associated with the mapping of the peptidesidentified by MS/MS analysis. Specifically, a set of 8 peptides wereisolated that mapped to 3 different regions on the CD44 molecule.Particularly, one peptide mapped to v8-v9 region which is unique toCD44E in addition to being present in the parent molecule.

FIG. 14 represents the sequence coverage obtained from mapping thepeptides obtained in the protein database. A set of 8 peptides wereobtained in all mapping the extracellular region, one in the variableregion and 4 in the cytoplasmic region of the CD44 molecule. Thehomology searches and mapping of peptides to CD44 variants indicate thatCD44R1 and CD44 R2 also express v8-v10 exons in the variable region.However, they lack a major portion of the cytoplasmic tail from the exon19. Therefore show homology only to 4 peptides out of 8 identified fromour analysis, hence do not fit into the criterion of Molecular weight/Piobserved from the antigen purified by immunoprecipitation. The predictedmolecular weight of 53.8 kDa for CD44E and the observed molecular weightand Pi proved to be an exact match. Therefore, the CD44 isoform that isthe possible antigen for VB1-008 is CD44E or the epithelial form, alsoreferred to as Isoform-3.

Example 7(c) Validation of VB1-008 Antigen

(1) Cell Surface Reactivity of Anti-CD44 and Anti-AFP by Flow Cytometry

The possibility of CD44 being the cognate antigen for VB1-008 has beenclearly established through immunopurification, gel-based analysis andMS analysis. Membrane preparations have been used in all the studiesperformed with VB1-008 based on the preliminary characterizationexperiments that clearly suggested the membrane localization of theantigen binding to VB1-008. To determine the orientation of the twocomponents of the antigen on the cell surface, reactivity was measuredby flow cytometry on a panel of cell lines, with VB1-008, anti-CD44,anti-AFP and anti-EGFR. Appropriate isotype-matched controls were alsoused in the study.

A panel of cell lines expressing different levels of VB1-008 Ag wasselected for comparative cell surface reactivity experiments.Approximately, 300,000 cells from each cell line were used and thefold-increase in median fluorescence of VB1-008/anti-CD44/anti-AFP wasmeasured and compared to the respective isotype-matched controls. Theantigen intensity column was a compilation of the signal intensityobserved on WB analysis for each cell line, probed with thecorresponding antibodies. The isotype-matched control for VB1-008 was4B5-IgG and the control for anti-CD44, anti-AFP and anti-EGFR were mouseIgG, since the latter three antibodies were mouse monoclonal antibodies.

As seen in Table 13, the rank order of the binding of anti-CD44 wassimilar to VB1-008. Anti-AFP did not show any detectable binding overthe isotype-matched control. Since anti-CD44 and anti-AFP were mousemonoclonal antibodies, anti-EGFR, a mouse monoclonal antibody was usedas a positive control. Not only was the rank order of bindingcomparable, anti-CD44 showed an enormous increase of over 48-foldcompared to the binding of VB1-008, suggesting the presence of a cognateantigen-antibody interaction. The antigen intensity as observed fromWestern blotting profiles also was comparable to the profile obtained byflow.

(2) 1D-PAGE/Western Blotting Analysis of Recombinant AFP

AFP is a serum glycoprotein that is available commercially as a 67 kDarecombinant molecule. This molecule was purchased from RDI laboratoriesand 0.3 μg of the pure protein, AFP and 0.3 μg of BSA wereelectrophoresed on SDS-PAGE, transferred to nitrocellulose membrane andprobed with VB1-008. As can be seen from FIG. 15A, positive reactivitywas observed indicating the presence of an epitope on AFP that isrecognized by VB1-008. Since AFP was one of the two identified proteinmolecules purified by immunoprecipitation with VB1-008 and identified byMS analysis, the current western blotting experiment proves the presenceof AFP in the immunopurified sample by VB1-008.

(3) Western Blot Analysis of VB1-008 Ag and Reactivity with Anti-AFP andAnti-CD44

2D-PAGE separation of the eluates from the VB1-008 immunoprecipitationreaction of MDA-MB-435S membranes revealed the presence of two distinctspots, “C” and “D”, in the Pi range of 5.1-5.4 and molecular weight 51+3kDa, and Pi range 5.2-5.5 and 50±3 kDa respectively. The two spots werevisualized when probed with VB1-008 as well. LC-MS/MS analysis of thesetwo spots revealed the identities of AFP and CD44, whose presence wasconfirmed even in the 110 kDa band seen under non-reducing conditions.Therefore, as a next step, the same conditions of immunopurificationwere repeated, resolved on 2D-PAGE, transferred to nitrocellulosemembranes and the Western blots were probed with anti-AFP and anti-CD44.The results are shown in FIG. 15B and FIG. 15C.

Each of the commercially available antibodies, anti-AFP and anti-CD44reacted specifically with the cognate spots identified by MS analysisfrom FIGS. 11A and 11B as spots “C” and “D” respectively. In FIGS. 15Band C, two spots around the same Pi, differing by 2-3 kDa were seeninteracting to anti-CD44, possibly due to some random loss of a fewamino acids as a processing by-product or due to the sensitivity ofanti-CD44 to recognize the presence of surrounding CD44 epitopes. Thepoint that needs to be emphasized is that the two spots that reactedwith VB1-008, identified to be AFP and CD44 have been visualized withthe respective antibodies at the appropriate positions of mass and Pi.

(4) Cross-Reactivity of AFP to CD44

In order to understand the relationship of AFP to CD44, an experimentwas designed to immunoprecipitate all CD44 isoforms, using anti-CD44.These proteins selectively purified were subjected to SDS-PAGE and WB.Three sets of identical experiments were carried out simultaneously.Western blots were probed with anti-CD44.

As can be seen in FIG. 16, AFP very strongly reacts with CD44 between115-200 kDa range when experimented under non-reducing conditions.VB1-008 reacts with CD44 as expected and is seen as a clean single bandat ˜110 kDa range as has been seen in previous cases. Therefore it ispossible that AFP is yet another co-purifying protein that possesses aninherent capacity to interact with CD44. As a result of being bound toCD44, it gets pulled down when immunopurified with VB1-008.

Discussion

Immunopurification experiments with VB1-008 showed a single specificband at ˜110 kDa under non-reducing conditions and a single 50+3 kDaband under reducing conditions of 1D-PAGE. In order to resolve proteinstacking possibilities and to determine the isoelectric point of theprotein, 2D-PAGE analysis was performed. Results from 2D-PAGE analysisshowed the presence of two spots at Pi=5.1-5.4 and 5.2-5.5 withmolecular weights of 51±3 kDa and 50±3 kDa, respectively. MS/MS analysisof the 2D spots recovered 32 and 8 peptides, spanning 54% and 28% ofeach protein identified, respectively. The two putative antigensidentified were CD44 isoform 3 and low molecular weight form ofalpha-fetoprotein.

Validation experiments were performed to confirm the presence of thesuggested antigens. SDS-PAGE/Western blot analysis of recombinant AFPmolecule probed with VB1-008 showed positive reactivity in the 67 kDarange as one strong single band, thus confirming the presence of AFP. Toconfirm the presence of CD44, the same panel of cells was tested usinganti-CD44 by flow cytometry. CD44 showed a dramatic increase in bindingcompared to VB1-008, also preserving the same rank order. AFP failed tobind to any of the cell lines tested. These results suggest that CD44 isthe cell surface antigen that is recognized by VB1-008. Also,immunopurification and subsequent MS/MS analysis clearly implicate theinvolvement of AFP.

CD44E as the VB1-008Ag

Protein identification was done with m/z measurements of trypticpeptides from VB1-008 Ag purified by immunoprecipitation. Thoroughsearches of the protein databases led to one perfect hit correspondingto a set of 8 peptides identified from the immunopurified VB1-008 Ag,pointing to CD44 isoform 3 also known as CD44E or the epithelial form.The molecular weight of the purified antigen, rules out the possibilityof both isoforms (1 and 2) as the antigen recognized by VB1-008 on thecells lines. Other isoforms such as isoform 2 which encodes all theexons except v1 or CD44v3, 8-10 could also be expected to react withVB1-008 but their molecular weight and/or pi are not consistent withthose observed for the VB1-008 cell surface antigen.

We show evidence for the occurrence of the predicted molecular weight ofthe CD44E or isoform 3 as 50±3 kDa on both 2D-PAGE, probed withanti-CD44 and on 1D-PAGE under reducing conditions of samplepreparation, which under non-reducing conditions was observed as 110±10kDa on 1D-PAGE and Western blot analysis. LC-MS/MS analysis of theproteins confirms the presence of CD44E.

Example 8 Epitope Mapping—Binding Experiments

As described above, immunoprecipitation and MS analysis have identifiedCD44E (isoform 3) as the VB1-008 antigen. CD44E differs from othersplice variants in having exons v8-v10 in between the conservedsequences, exons 1-5 and 16-20. Peptides were then synthesized from theunique region of CD44E (i.e., the amino acid sequence that spans theexon 5-v8 junction) in order to identify the reactive epitope ofVB1-008. A peptide of the same length taken from the C-terminal regionof CD44E was used the negative control.

Methods and Reagents

Peptides from the Unique Region of CD44E:

Synthetic peptides spanning the exon 5-V8 junction of CD44E were orderedfrom Global peptide services, LLC. The amino acid sequence (17 aa) fromCD44E spans a length of 6 amino acids from exon 5 and 11 amino acidsfrom the unique peptide of the v8 region. The highlighted portion ofFIG. 18A represents the stretch of 17 amino acids which has been splitinto 3 peptides, and the negative control peptide sequence is ashighlighted in the C-terminal region of the protein.

The amino acid sequence of each peptide is as follows:

(SEQ ID NO: 26) Peptide 1: Biotin-STDRIPATNMD-1445.2 amu (SEQ ID NO: 27)Peptide 2: Biotin-RIPATNMDSSH-1453.27 amu (SEQ ID NO: 28)Peptide 3: Biotin-ATNMDSSHSIT-1387.58 amu (SEQ ID NO: 29)Negative:  Biotin-AVEDRKPSGLN-1410.19 amuSolubilizing Peptides:

All peptides were solubilized in PBS. The pH of the solution wasadjusted with 0.01 N HCl or 0.01 N NaOH if any difficulty in solubilitywas observed. The peptide was stored in stock solutions (1000 nM) at−20° C.

Coating the Peptides on an ELISA Plate:

Peptide solutions were diluted 1-in-100 with Hank's buffered salinesolution (HBSS) containing 0.5% formaldehyde. 10Q^iL of diluted peptidesolution was distributed to each well in a 96-well plate. The plateswere incubated at room temperature for 1 hour. The supernatant wasremoved and the plates were placed uncovered in a 37° C. incubator for16-18 hours. The peptide-coated plates were placed in plastic bags andstored at 2-8° C. until required.

Alternatively, the peptides were diluted in carbonate/bicarbonate bufferpH 9.6 and coated on the plates. All the other steps with the exceptionof a change in the coating buffer were the same.

Binding of VB1-008 to the Peptide-Coated ELISA Plates:

VB1-008 binding to immobilized peptides was performed according to SOP2.1.19 and SOP 2.2.7:

Following overnight incubation of the peptide-coated plates, 300 μL ofwash buffer (PBS containing 0.5% Tween20) was manually added to eachplate, with the help of a repeator pipette equipped with an 8-channeladaptor. The contents of the plates were discarded; the plates wereinverted and patted on 3-4 inches of paper towel to remove excessliquid. The above steps were repeated two more times.

Blocking:

The peptide-coated plates were blocked with 300 μL/well with blockingbuffer (PBS containing 1% BSA). The plates were incubated for 30-60minutes at room temperature. The block buffer was discarded after theincubation.

Binding:

Aliquots equivalent to 75 μg/mL of VB1-008 were added to each of thewells and incubated at 37° C. for two hours. The plates were washed aspreviously described with the wash buffer (PBS containing 0.5% Tween20). The plates were incubated with 1:6000 dilution of anti-humanIgG-HRP for one hour at room temperature. The plates were washed aspreviously described. 100 μL of TMB substrate (TMB peroxidase substrateKPL cat #50-76-00) was added to each well and incubated for 5-10 minutesin the dark. The reaction was terminated by adding 100 μL of 1Mphosphoric acid to each well. The optical density was measured at 450 nmusing an ELISA plate reader.

Alternatively, ELISA plates were coated with 100 μg/mL of VB1-008,according to the SOP 2.1.111, and binding of the biotinylated peptidesto VB1-008 were assayed according to SOP 2.1.41 for the detection ofbiotinylated probes.

Results

Screening of synthetic peptides from the unique region of CD44E (i.e.,the amino acid sequence that spans the exon 5-v8 junction), revealedthat Peptide 3 showed the strongest binding, followed by peptide 2 whichdemonstrated 50-60% of the binding observed with Peptide 3. A peptide ofthe same length taken from the C-terminal region of CD44E used asnegative control did not show any reactivity as was the case withPeptide 1. Reactivity of VB1-008 with peptide 3 demonstrated that thisregion of CD44E contains the reactive epitope of VB1-008. See FIG. 18B.

Example 9 Epitope Mapping—Competition Experiments

The competing efficiency of the peptides for VB1-008 binding was thenassayed.

Methods and Reagents

Growth and Maintenance of Tumor Cell Lines:

Cell lines that are VB1-008-positive, i.e., MDA-MB-435S were culturedand maintained according to ATCC guidelines.

Synthetic Peptides:

All peptides were solubilized in PBS and stored at 1.428 mM (2 mg/mL)and as 100 μM solutions at −20° C.

Competition Assay:

VB1-008 (75 μg/mL)—0.5 μM concentration, was used as the non-competedcontrol. Molar excesses, i.e., 20×, 40×, 100× and 200× of peptides wereused to compete with VB1-008. The peptides A/B 1-008 mixtures wereincubated on ice for 10 minutes prior to binding by flow. 4B5-IgG wasused as the Isotype-matched control and anti-EGFR was used as theunrelated antibody. These two antibodies were processed exactly the sameas VB1-008.

Binding of VB1-008:

The binding of VB1-008, along with the anti-EGFR and 4B5-IgG antibodiesto MDA-MB435S cells was assessed by flow cytometry; and was performedaccording to the optimized protocol previously described. Cells treatedwith peptides and those that were untreated were processed similarly.

Results

As seen in FIG. 19A, peptide 1 did not compete with VB1-008 binding toMDA-MB435S, peptide 2 competed at 60% efficiency with VB1-008 binding toMDA-MB435S and peptide 3 competed at 96% efficiency with VB1-008 bindingto MDA-MB435S. The control showed no competition to VB1-008.

FIG. 19B shows the results of the isotype-matched control. None of thepeptides or controls compete with anti-EGFR for binding.

Example 10 Cytotoxicity of VB1-008 Immunotoxin

Methods and Reagents

The VB6-008 construct, comprising VB1-008 attached to a modifiedbouganin was constructed using the methods disclosed inPCT/CA2005/000410 and U.S. patent application Ser. No. 11/084,080.

A dicistronic expression unit was generated comprising the VH-CH domainof VB1-008 linked to modified bouganin using a furin-sensitive linkerimmediately followed by the VL-CL of VB1-008 domain. Both the VH and VLwere preceded by a PelB leader sequence (See FIGS. 26 and 27). Thedicistronic unit was cloned into the pING3302 Xoma vector and was underthe control of the arabinose-inducible araBAD promoter. The presence ofthe PelB leader sequence, adjacent to VH-CH Bouganin and VL-CL, willresult in secretion of the proteins into the periplasmic space where thereducing environment will allow the formation of the disulphide bridgebetween the two constant domains. Ultimately, the Fab-bouganin fusionprotein will be secreted into the culture supernatant. A histidineaffinity tag, placed at the N-terminal of the VL-CL enables theFab-bouganin protein to be purified using a Ni²⁺ chelating capturemethod. The VH fragment of VB6-008 (395 bp) was amplified with thefollowing primers and cloned into PelB-VB6-011-F-boug gamma cassetteusing Pvull and Nhel restriction sites.

5¹ Pvull-QVQL (SEQ ID NO: 30) 5′ATG GCG CAG GTG CAG CTG CAG GAG TTG GGT CCA 3′ VB4-008-Nhel(SEQ ID NO: 31) 5′ CGA TGG GCC CTT GGT GGA GGC GCT AGC GAC AGT GACCAT TGT CCC

VB1-008 light chain is a lambda and since the lambda CL domain containsa Spel restriction site, a different restriction site was used toassemble VB6-008. Therefore, in the 5′ end of the VB6-008 light chainfragment, the HindIII restriction site (in bouganin) was used toassemble the final construct into pSP73 plasmid (See FIG. 27). Norestriction site was found around the VL-CL junction therefore the VL-CLof each clones was obtained by the Splice Overlapping Extension PCRapproach. The following primers were used along with D-bouganin 156,PelB signal and cDNA of VB1-008 hybridoma as templates:

HindIII-boug-PelB-VB6-008 lambda was assembled by the Splice OverlappingExtension Polymerase Chain Reaction method using the following primers:

5′ Furin Linker D-bouganin (SEQ ID NO: 32) 5′CAC AGG CAG CCC AGA GGC TGG GAG CAG CTC TAC AAC ACC GTG TCA TTT AAC CTT3′ 008-PelB (SEQ ID NO: 33) 5′CGT TCC ATA GAC CTG CAG TCT AGA GTC GAC TCA CTA TTT GGA GCT TTT AAA CTT5′ PelB-Sall (SEQ ID NO: 34) 5′AAG TTT AAA AGC TCC AAA TAG TGA TCT AGA GTC GACCTG CAG GTC TAT GGA ACG ATA AAT 3′ 008-VL CL (SEQ ID NO: 35) 5′CAC TGA GGG TGG CTG AGT CAG CTC ATA GTG ATG GTG GTA GTG AGT 5′ 008-VL CL(SEQ ID NO: 36) 5′ CAT CAC CAT CAC CAT CAC TAT GAG CTG ACT CAG CCACCC TCA GTG 3′ 008 CL STOP (SEQ ID NO: 37) 5′CTC GAG TCA CTA TGA ACA TTC TGT AGG GGC CAC TGT CTT CTC CAC

A three-step Splice Overlapping Extension PCR approach was undertakenusing all 6 primers listed above for amplification.

Step 1

Primers 1 and 2 was used to amplify bouganin containing a portion of thePelB promoter (820 bp) in the 3′ end. In a second PCR reaction, primers3 and 4 was used to amplify the PelB containing in the 3′ end a His tagand a portion of VB6-008 VL (179 bp). In a third PCR reaction, primers 5and 6 was used to amplify the VB6-008 lambda chain with two stop codonsand the Xhol site (666 bp) in the 3′ end.

Step 2

In the second PCR reaction, primers 1 and 6 was used with VI from eachPCR product to produce the Hindlll-bouganin-PelB-VB6-008 lambda chain(1591 bp).

Electrophoresis on a 1% agarose gel was used to separate the amplifiedPCR products. The bands of interest was excised and purified using aQiaquick gel extraction kit, cloned into the TOPO pCR 2.1 cloning vectorand sequenced using the 373 DNA sequencer.

The PCR product was purified and sequenced. A verified clone wasdigested with Hindlll and Xhol and ligated into thePelB-VB4-008-F-boug/pSP73 previously digested with the correspondingenzymes (FIG. 27). The VB6-008 fragment was then be digested with EcoRIand Xhol and cloned into the plNG3302 expression vector and transformedinto E104 cells.

E104 cells were propagated in 30 mL of TB media (1% innoculum) in a 250mL shake flask at 37° C., shaken at 225 rpm for approximately 5 hoursuntil the optical density (O.D. 600 nm) reached 2. At this time, theculture was induced with a final concentration of 0.1% L-(+) arabinosefor 16 hours and incubated at 25° C. Subsequently, the cell pellet andsupernatant was collected by centrifugation at 14000 rpm for 5 minutes.Both the cell pellet and supernatant was analyzed by Western blot usingan anti-His (Amersham Biosciences 27-4710-01) and an anti-human kappalight chain (Sigma A-7164) or anti-human lambda light chain (SigmaA-5175) under reducing and non-reducing conditions to confirm thepresence and size of the immunotoxin. A Research Cell Bank of the clonewith the highest expression level was made and three independent vialswill be tested for induction at a scale of 500 mL TB in 2 L shakeflasks. Every 6 hours, the cell pellet and supernatant was isolated andWestern blot analysis was used to indicate the optimal post-inductiontime for harvesting.

Flow cytometry was used to demonstrate that the purified VB6immunotoxins retain the binding specificity of their respective parentantibody using antigen positive and negative cell lines. Binding will bedetected using a mouse anti-His monoclonal antibody (AmershamBiosciences 27-4710-01). The specificity of the binding was assessed bycompetition assay. Briefly, the VB6-immunotoxin (at a fixedconcentration) and the corresponding VB1 antibody or an isotype-matchedcontrol antibody (at varying concentrations) was incubatedsimultaneously with antigen positive cells. Binding was detected using amouse anti-His monoclonal antibody. Decreased binding using the anti-Hismonoclonal antibody indicated that the VB6 immunotoxins and thecorresponding VB1 antibody bind to the same antigen. It is expected thatthe level of binding of the VB6 immunotoxins will not be altered in thepresence of the isotype-matched control antibody. The functionalaffinity of the VB6 immunotoxins was calculated with a titration curveusing an antigen positive cell line. An MTS assay was used to measurethe IC50 of each VB6 immunotoxin using antigen positive and negativecell lines. VB6-4B5 was used as a negative control. The specificity ofthe cytotoxicity was measured by the difference in IC50 between the VB6immunotoxins and VB6-4B5.

Results

An immunoconjugate (VB6-008) comprising VB1-008 attached to a modifiedbouganin was constructed. The nucleotide sequence of the immunoconjugateis depicted in FIG. 20 (SEQ ID NO:11). The amino acid sequence of theimmunoconjugate is depicted in FIG. 21 (SEQ ID NO:12). FIG. 22 shows thecomplete VB6-008 construct. FIG. 23 shows VB6-008 unit #1, whichincludes PelB-VH-CH-Furin-De-Bouganin. FIG. 24 shows VB6-008 unit #2,which consists of PelB-VL-CL.

The cytotoxicity of VB6-008 was assessed in vitro against theantigen-positive cells, MB-435SC. Colo-320 was used as the negativecontrol. The cells were incubated with VB8-008 ranging from 1000 to 1 nmand after 5 days of incubation variability was measured. As can be seen,in FIG. 25, the VB6-008 immunoconjugate significantly killed theantigen-positive cells as compared to the negative control.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

TABLE 1 CDR Sequences CDR Sequences VB1-008 L-chain H-chain CDR1SGDNLGNKYVC SEQ ID GDEYYWS SEQ ID NO: 1 NO: 4 CDR2 EDTKRPS SEQ IDYMSYRGSSYYSPSLQS SEQ ID NO: 2 NO: 5 CDR3 QAWDSRTEI SEQ ID KYCGGDCRSGFDISEQ ID NO: 3 NO: 6

TABLE 2 Comparison of normal and tumor cell surface binding with VB1-008Clinical Indication Representative Tumor Cell lines N¹ MF* Relative RankBreast MCF-7, MDA-MB-231, MDA-MB-435S 3 17.2 1 Lung A-549, NCI-H460,NCI-H69 3 16.1 2 Melanoma A-375, SK-MEL-5^(a,b), SK-MEL-28^(a) 3 15.6 3Prostate DU-145^(a,b,f), PC-3^(a,b,g), LNCaP^(a,b,g) 3 14.2 4 OvarianSK-OV-3^(a), OVCar-3 2 10.8 5 Kidney Caki-1^(a), A498^(a), ACHN^(a) 310.5 6 Liver SK-HEP-1, Hep-G2 2 8.3 7 Rectum SW837, NCI-H630 2 7.5 8Colon HT-29^(a), SW480, WiDr 3 7.2 9 Cervix HeLa, C-41, C33A 3 4.4 10Stomach AGS, NCI-N-87, KATO III 3 4.0 11 Bladder UM-UC-3, T24 2 3.9 12Endometrium RL-95-2, HEC-1-A 2 3.9 12 Pancreas PANC-1, BxPC-3, MIAPaCa-2 3 3.8 14 Head & Neck SCC-15, SCC-25 2 2.9 15 Normal Cell TypeCell Line Tumor:normal Kidney HRE 1 6.1 1.7 Lung NHLF 1 5.6 72.9Endothelial HUVEC 1 1.6 N/A Breast HMEC 1 2.4 7.2 Prostate PrEC 1 4.03.6 ¹N indicates the number of cell lines tested per indication. ²MF:Values indicate the mean calculated from the sum of the mean foldincrease in median fluorescence over the control antibody from all celllines in each indication. A zero value indicates no measurablereactivity relative to the control antibody. ^(a)Indicates orthotopicmodels offered by AntiCancer Inc. ^(b)Indicates cell lines available asGFP (green fluorescent protein)-transfectants. ^(c)Her2/neu⁻, ER⁺.^(d)Her2/neu⁻, ER⁻, p53^(wt), ras^(wt). ^(e)Her2/neu⁻, ER⁻, p53^(mt),ras^(wt). Androgen-responsive. ^(g)Androgen-unresponsive. N/A, notapplicable. The mean-fold increase (MF) is used to calculate thetumor:normal ratio.

TABLE 3 LD Array of Critical Normal Tissue for VB1-008 Tissue MembraneStaining Score Range* Brain None (0/2) 0 Colon None (0/5) 0 Heart None(0/5) 0 Kidney 2/3 0-1 (10%) Liver None (0/5) 0 Lung None (0/5) 0Pancreas 1/5 1 (30%) Stomach 1/5 1 (70%) *Scoring was evaluated on a0-3+ scale, with 0 = no staining and trace being less than 1+ butgreater than 0. Grades 1+ to 3+ represent increased intensity ofstaining, with 3+ being strong, dark brown staining. In general, asingle specimen of 6 different patients was screened. Where fewer than 6patients were screened indicates cores were either missing or were notrepresentative of the tissue to be stained. Values in parenthesesindicate the percentage of cells stained in the scored range.

TABLE 4 HD Normal TMA for VB1-008 Membrane Tissue Staining Score Range*Adrenal None (0/2) 0 Aorta None (0/5) 0 Artery None (0/5) 0 Bladder None(0/5) 0 Brain None (0/5) 0 Breast None (0/5) 0 Fallopian 3/4 1-2(30-60%) LN None (0/3) 0 Muscle None (0/4) 0 Ovary None (0/5) 0Pituitary None (0/5) 0 Placenta None (0/4) 0 Prostate 4/5 0-1 (10-20%)Skin ND Spinal cord None (0/1) 0 Spleen None (0/2) 0 Testis 3/5 1-2(95%) Thymus None (0/1) 0 Thyroid None (0/5) 0 Ureter 1/2 Uterus None(0/5) 0 *Scoring was evaluated on a 0-3+ scale, with 0 = no staining andtrace being less than 1+ but greater than 0. Grades 1+ to 3+ representincreased intensity of staining, with 3+ being strong, dark brownstaining. In general, 2 specimens of 8 different patients were screened.Where fewer than 8 patients were screened indicates cores were eithermissing or were not representative of the tissue to be stained. Valuesin parentheses indicate the percentage of cells stained in the scoredrange.

TABLE 5 HD Tumor TMA for VB1-008 Membrane Score Tissue Staining Range*Bladder 6/6 1-2 (100%) Breast 6/7 1-2 (100%) Cervix 2/7   1 (100%) Colon3/3 1-2 (100%) Kidney 5/8 1-2 (100%) Liver 5/7 1-2 (100%) Lung 1/8   1(100%) Ovary 6/7 1-2 (100%) Pancreas 4/7   1 (100%) Prostate 5/5 1-2(100%) Rectum 4/6 1-2 (100%) Skin 1/4   1 (100%) Stomach 4/5 1-2 (100%)Uterus 8/8 1-2 (100%) Head & Neck 4/8   1 (100%) *Scoring was evaluatedon a 0-3+ scale, with 0 = no staining and trace being less than 1+ butgreater than 0. Grades 1+ to 3+ represent increased intensity ofstaining, with 3+ being strong, dark brown staining. In general, 2specimens of 8 different proteins were screened. Where fewer than 8proteins were screened indicates cores were either missing or were notrepresentative of the tissue to be stained. Head & neck cancers includedcarcinomas of the throat, lip, larynx, mouth, tonsil, and gingivalsurface. Values in parentheses indicate the percentage of cells stainedin the scored range.

TABLE 6 Flow cytometry assessment of antibody binding as a function oftime and temperature Incubation Fold- % Time Median increase Re- (min)at Fluorescence in duction MAb ID Antibodies¹ 37° C. (MF) MF² in MF³VB1-008 17P2/C12 —⁴ 134.0 ± 11   31.7 —  60 57.0 ± 1.0 13.5 57.5 12050.7 ± 1.1 12.0 62.2 Non- MA-103 — 536.1 ± 31.3 112.8 — Internalizing120 535.5 ± 16.8 113.0 — Control Internalizing 5E9 — 246 ± 11 60.0 —Control  60 53.5 ± 1.5 13.0 78.3 120 48 ± 4 11.7 80.5 ¹A representativeexperiment is shown. ²MF increase above the negative control, mousemyeloma IgG or human IgG (4B5). ³Percent reduction of MF from thecell-surface of tumor cells. ⁴(—) cells incubated on ice for 120minutes.

TABLE 7 Increase in median fluorescence for VB1-008 over anisotype-matched control for each cell line used in the study Cell lineMF* A-375 13.3 MDA-MB-435S 15.8 MDA-MB-231 14.2 MCF-7 4.67 PANC-1 8.3DAUDI 1.1 RAMOS 1.3

TABLE 8 Summary of the antigens purified Sample preparation Flow Cellline reduced non-reduced results intensity A-375 50 ± 2 kDa 100 ± 5 kDa11.08 +++ MB435S 50 + 2 kDa 100 ± 5 kDa 15.8 ++++ MB231 50 + 2 kDa 100 ±5 kDa 14.2 ++ MCF-7 50 + 2 kDa 100 ± 5 kDa 4.63 + PANC-1 — — 8.95 −DAUDI — — 1 − RAMOS 50 + 2 kDa 100 ± 5 kDa 1.1 +++

TABLE 9A Summary of the proteins identified by LC-MS/MS from 2D spot -‘C’ 2D Spot ‘C’ - 48.8 kDa from MDA-MB-435S Match to Accession # ProteinID Mw/Pi Peptides 2DE gi|4501989 alpha-fetoprotein 68813/5.2 30 84✓[Homo sapiens](AFP) gi|231315 alpha-1proteinase 39099/5.27 7 C inhibitorgi|224224 alpha-1 antitrypsin 46731/4.35 6 C C - Co-purifyingcontaminant; X - does not match Pi and/or Mw observed; ✓ = matches Piand Mw

TABLE 9B Summary of the proteins identified by LC-MS/MS from 2D spot -‘D’ 2D Spot ‘D’ - 45 + 2 kDa Match to Accession # Protein ID Mw/PIPeptides 2DE gi|105583 cell adhesion molecule 53585/5.4 3 ✓ CD44 - humangi|87056 nucleolin-related 77453/4.5 3 X protein - human gi|2804273alpha-actinin 4 102661/5.27 5 C [Homo sapiens] gi|34862435 ER protein99/integrin  92713/4.72 2 X gi|71620 actin-beta - bovine  41786/5.22 1 cC - o-purifying contaminant; X - does not match Pi and Mw observed; ✓ =matches pi and Mw within acceptable range

TABLE 10 Summary of the proteins identified by LC-MS/MS from proteinband ‘E’ Protein band ‘E’ - 110 kDa band from VB1-008 IP (non-reducingconditions) Match to Accession # Protein ID Mw/PI Peptides 2DE gi14501989 alpha-fetoprotein 68813/5.2 16 ✓ [Homo sapiens] (AFP) Gi|105583cell adhesion 53585/5.4 8 V molecule CD44 - human gi|20177936 heat shockprotein 81912/4.77 10 X Hsp90-beta[Hsp 84] gi|34862435 Alpha-actinin92713/4.72 2 C gi|71620 actin-beta - bovine 41786/5.22 5 C gi|55408vimentin 54418/5.01 3 C [Mus musculus] C - Co-purifying contaminant; X -does not match Pi and Mw observed; ✓ = matches pi and Mw withinacceptable range

TABLE 11 A List of peptides recovered from MS/MS for AFP PeptideSEQ ID NO YGHSDCCSQSEEGR 46 HNCFLAHK 47 FIYEIAR 48 HPFLYAPTILLWAAR 49IIPSCCK 50 AENAVECFQTK 51 ESSLLNQHACAVMK 52 TFQAITVTK 53 LSQKFTK 54LVLDVAHVHEHCCR 55 GDVLDCLQDGEK 56 IMSYICSQQDTLSNK 57GQCIIHAENDEKPEGLSPNLNR 58 FLGDRDFNQFSSGEK 59 DFNQFSSGEK 60DFNQFSSGEKNIFLASFVHEYSR 61 NIFLASFVHEYSR 62 RHPQLAVSVILR 63 HPQLAVSVILR64 GYQELLEK 65 YIQESQALAKR 66 RSCGLFQK 67 LGEYYLQNAFLVAYTKK 68KAPQLTSSELMAITR 69 APQLTSSELMAITR 70 MAATAATCCQLSEDKLLACGEGAADII 71IGHLCIR LLACGEGAADIIIGHLCIR 72 DLCQAQGVALQTMKQEFLINLVK 73 QEFLINLVK 74QKPQITEEQLEAVIADFSGLLEK 75

TABLE 11B List of peptides recovered from MS analysis ofimmunopurified CD44 NLQNVDMK-Exon 20 (SEQ ID NO: 38)YVQKGEYR-Exon 5 (SEQ ID NO: 39) KPSGLNGEASK-Exon 20 (SEQ ID NO: 40)YGFIEGHWIPR-Exon 3 (SEQ ID NO: 41) TEAADLCK-Exon 2 (SEQ ID NO: 42)LVINSGNGAVEDR-Exon 19 (SEQ ID NO: 43)ESSETPDQFMTADETR-Exon 20 (SEQ ID NO: 44)TGPLSMTTQQSNSQSFSTSHEGLEED-Exon v8-v9 (SEQ ID NO: 45)

We claim:
 1. An isolated nucleic acid sequence comprising the cDNAsequence as set forth in SEQ ID NO:8 encoding for a light chain variableregion.
 2. An isolated nucleic acid sequence comprising the cDNAsequence as set forth in SEQ ID NO:10 encoding for a heavy chainvariable region.
 3. An isolated cDNA molecule encoding a binding proteincomprising: a light chain complementarity determining region 1 (CDR1)comprising the amino acid sequence of SEQ ID NO:1; a light chaincomplementarity determining region 2 (CDR2) comprising the amino acidsequence SEQ ID NO:2; a light chain complementarity determining region 3(CDR3) comprising the amino acid sequence SEQ ID NO:3; a heavy chaincomplementarity determining region 1 (CDR1) comprising the amino acidsequence SEQ ID NO:4; a heavy chain complementarity determining region 2(CDR2) comprising the amino acid sequence SEQ ID NO:5; and a heavy chaincomplementarity determining region 3 (CDR3) comprising the amino acidsequence SEQ ID NO:6.
 4. The isolated cDNA molecule of claim 3, whereinthe binding protein is an antibody.
 5. The isolated cDNA molecule ofclaim 4, wherein the antibody is an antibody fragment.
 6. The isolatedcDNA molecule of claim 5, wherein the antibody fragment is a Fab, Fab′,F(ab′)2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, andmultimers thereof or bispecific antibody fragments.
 7. A recombinantexpression vector comprising the cDNA molecule of claim
 3. 8. Anisolated nucleic acid molecule encoding an immunoconjugate comprising acancer therapeutic that is cytotoxic, cytostatic or reduces the abilityof the cancer cells to divide or metastasize, wherein the bindingprotein is an antibody comprising a light chain complementaritydetermining region 1 (CDR1) comprising the amino acid sequence of SEQ IDNO:1; a light chain complementarity determining region 2 (CDR2)comprising the amino acid sequence SEQ ID NO:2; a light chaincomplementarity determining region 3 (CDR3) comprising the amino acidsequence SEQ ID NO:3; a heavy chain complementarity determining region 1(CDR1) comprising the amino acid sequence SEQ ID NO:4; a heavy chaincomplementarity determining region 2 (CDR2) comprising the amino acidsequence SEQ ID NO:5; and a heavy chain complementarity determiningregion 3 (CDR3) comprising the amino acid sequence SEQ ID NO:6.
 9. Theisolated nucleic acid molecule of claim 8, wherein the cancertherapeutic is a toxin.
 10. The isolated nucleic acid molecule of claim9, wherein the toxin is a ribosome-inactivating polypeptide selectedfrom the group consisting of gelonin, bouganin, saporin, ricin, ricin Achain, bryodin, diphtheria, restrictocin and Pseudomonas exotoxin A. 11.The isolated nucleic acid molecule of claim 10, wherein the toxin ismodified bouganin as set forth in SEQ ID NO:
 17. 12. The isolatednucleic acid molecule of claim 10, wherein the toxin is a truncated forof Pseudomonas exotoxin A that consists of amino acids 252-608.
 13. Theisolated nucleic acid molecule of claim 8 comprising a nucleotidesequence of SEQ ID NO: 84 and SEQ ID NO:
 86. 14. The isolated nucleicacid of claim 8 encoding the amino acid sequence of SEQ ID NO: 12 and13.
 15. A recombinant expression vector comprising the nucleic acidmolecule of claim
 8. 16. A recombinant expression vector according toclaim 15 comprising the nucleic acid molecule according to SEQ ID NO: 84and SEQ ID NO: 86.